WorldWideScience

Sample records for bacterial cell biotherapeutics

  1. Investigation of Genipin Cross-Linked Microcapsule for Oral Delivery of Live Bacterial Cells and Other Biotherapeutics: Preparation and In Vitro Analysis in Simulated Human Gastrointestinal Model

    Directory of Open Access Journals (Sweden)

    Hongmei Chen

    2010-01-01

    Full Text Available Oral therapy utilizing engineered microorganisms has shown promise in the treatment of many diseases. By microencapsulation, viable cells can overcome the harsh gastrointestinal (GI environment and secrete needed therapeutics into the gut. These engineered cells should be encased without escaping into the GI tract for safety concerns, thus robust microcapsule membrane is requisite. This paper examined the GI performance of a novel microcapsule membrane using a dynamic simulated human GI model. Results showed that the genipin cross-linked alginate-chitosan (GCAC microcapsules possessed strong resistance to structural disintegration in the simulated GI environment. Leakage of encapsulated high molecular weight dextran, a model material to be protected during the simulated GI transit, was negligible over 72 h of exposure, in contrast to considerable leakage of dextran from the non-cross-linked counterparts. These microcapsules did not alter the microflora and enzymatic activities in the simulated human colonic media. This study suggested the potential of the GCAC microcapsules for oral delivery of live microorganisms and other biotherapeutics.

  2. Mast cells in bacterial infections

    OpenAIRE

    Rönnberg, Elin

    2014-01-01

    Mast cells are implicated in immunity towards bacterial infection, but the molecular mechanisms by which mast cells contribute to the host response are only partially understood. Previous studies have examined how mast cells react to purified bacterial cell wall components, such as peptidoglycan and lipopolysaccharide. To investigate how mast cells react to live bacteria we co-cultured mast cells and the gram-positive bacteria Streptococcus equi (S. equi) and Staphylococcus aureus (S. aureus)...

  3. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  4. Bacterial cell division proteins as antibiotic targets

    NARCIS (Netherlands)

    T. den Blaauwen; J.M. Andreu; O. Monasterio

    2014-01-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of protein

  5. Bacterial Probiotic Modulation of Dendritic Cells

    OpenAIRE

    Drakes, Maureen; Blanchard, Thomas; Czinn, Steven

    2004-01-01

    Intestinal dendritic cells are continually exposed to ingested microorganisms and high concentrations of endogenous bacterial flora. These cells can be activated by infectious agents and other stimuli to induce T-cell responses and to produce chemokines which recruit other cells to the local environment. Bacterial probiotics are of increasing use against intestinal disorders such as inflammatory bowel disease. They act as nonpathogenic stimuli within the gut to regain immunologic quiescence. ...

  6. Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO-cell derived biotherapeutics.

    Science.gov (United States)

    Strauss, Daniel M; Lute, Scott; Tebaykina, Zinaida; Frey, Douglas D; Ho, Cintia; Blank, Gregory S; Brorson, Kurt; Chen, Qi; Yang, Bin

    2009-10-01

    During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

  7. Antibiotic-Free Selection in Biotherapeutics: Now and Forever

    Directory of Open Access Journals (Sweden)

    Charlotte Mignon

    2015-04-01

    Full Text Available The continuously improving sophistication of molecular engineering techniques gives access to novel classes of bio-therapeutics and new challenges for their production in full respect of the strengthening regulations. Among these biologic agents are DNA based vaccines or gene therapy products and to a lesser extent genetically engineered live vaccines or delivery vehicles. The use of antibiotic-based selection, frequently associated with genetic manipulation of microorganism is currently undergoing a profound metamorphosis with the implementation and diversification of alternative selection means. This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the bio-therapeutic world.

  8. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

    Full Text Available Micropatterning of bacteria using aqueous two phase system (ATPS enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

  9. Current development in regulation of similar biotherapeutic products in Brazil.

    Science.gov (United States)

    Castanheira, Laura Gomes; Barbano, Dirceu Brás Aparecido; Rech, Norberto

    2011-09-01

    Because of the recent expiry of a large number of patents on the originator biological products, interest in the production and marketing of similar biotherapeutic products in Brazil has been increasing. The national producers have significant interest in this market and have been making a large amount of investments in these kinds of products. Since biotherapeutic products consume a large amount of the government health budget, the Brazilian government also has a big interest in the possibility that more affordable biotherapeutic products could be introduced into the market to improve access, but always is concerned with the quality, safety and efficacy of these products Accordingly, it was necessary to review the biological product regulations in Brazil and to establish specific pathways to license similar biotherapeutic products. The new Brazilian regulations, Resolution no. 55/2010, are based on different regulations and guidelines from around the world, including the WHO SBP Guidelines. They follow the same scientific principles as the WHO Guidelines but also have some differences which are due to specific country needs. PMID:21868247

  10. Similar biotherapeutic products in Latin America. Regulation and opportunities for patients with autoimmune diseases

    OpenAIRE

    Desanvicente-Celis Z; Caro-Moreno J; Enciso-Zuluaga M; Anaya JM

    2013-01-01

    Zayrho Desanvicente-Celis, Julian Caro-Moreno, Mateo Enciso-Zuluaga, Juan-Manuel AnayaCenter for Autoimmune Diseases Research (CREA), Universidad del Rosario, Bogotá, ColombiaAbstract: Biotherapeutic products have revolutionized medicine, changing the way we can treat some chronic diseases, such as autoimmune diseases. The patent expiry and the high costs of reference biotherapeutic products, among other factors, have promoted interest in similar biotherapeutic products (SBPs), also kn...

  11. Similar biotherapeutic products in Latin America. Regulation and opportunities for patients with autoimmune diseases

    OpenAIRE

    Anaya, Juan-Manuel

    2013-01-01

    Zayrho Desanvicente-Celis, Julian Caro-Moreno, Mateo Enciso-Zuluaga, Juan-Manuel AnayaCenter for Autoimmune Diseases Research (CREA), Universidad del Rosario, Bogotá, ColombiaAbstract: Biotherapeutic products have revolutionized medicine, changing the way we can treat some chronic diseases, such as autoimmune diseases. The patent expiry and the high costs of reference biotherapeutic products, among other factors, have promoted interest in similar biotherapeutic products (SBPs), als...

  12. Bacterial cell curvature through mechanical control of cell growth

    DEFF Research Database (Denmark)

    Cabeen, M.; Charbon, Godefroid; Vollmer, W.;

    2009-01-01

    The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure...

  13. Mapping the Pareto optimal design space for a functionally deimmunized biotherapeutic candidate.

    Science.gov (United States)

    Salvat, Regina S; Parker, Andrew S; Choi, Yoonjoo; Bailey-Kellogg, Chris; Griswold, Karl E

    2015-01-01

    The immunogenicity of biotherapeutics can bottleneck development pipelines and poses a barrier to widespread clinical application. As a result, there is a growing need for improved deimmunization technologies. We have recently described algorithms that simultaneously optimize proteins for both reduced T cell epitope content and high-level function. In silico analysis of this dual objective design space reveals that there is no single global optimum with respect to protein deimmunization. Instead, mutagenic epitope deletion yields a spectrum of designs that exhibit tradeoffs between immunogenic potential and molecular function. The leading edge of this design space is the Pareto frontier, i.e. the undominated variants for which no other single design exhibits better performance in both criteria. Here, the Pareto frontier of a therapeutic enzyme has been designed, constructed, and evaluated experimentally. Various measures of protein performance were found to map a functional sequence space that correlated well with computational predictions. These results represent the first systematic and rigorous assessment of the functional penalty that must be paid for pursuing progressively more deimmunized biotherapeutic candidates. Given this capacity to rapidly assess and design for tradeoffs between protein immunogenicity and functionality, these algorithms may prove useful in augmenting, accelerating, and de-risking experimental deimmunization efforts.

  14. One bacterial cell, one complete genome.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    Full Text Available While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA. Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs, indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  15. One Bacterial Cell, One Complete Genome

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  16. Metabolic Responses of Bacterial Cells to Immobilization

    Directory of Open Access Journals (Sweden)

    Joanna Żur

    2016-07-01

    Full Text Available In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability.

  17. Metabolic Responses of Bacterial Cells to Immobilization.

    Science.gov (United States)

    Żur, Joanna; Wojcieszyńska, Danuta; Guzik, Urszula

    2016-01-01

    In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability. PMID:27455220

  18. A gastrointestinal anti-infectious biotherapeutic agent: the heat-treated Lactobacillus LB

    Science.gov (United States)

    Liévin-Le Moal, Vanessa

    2016-01-01

    Experimental in vitro and in vivo studies support the hypothesis that heat-treated, lyophilized Lactobacillus acidophilus LB cells and concentrated, neutralized spent culture medium conserve the variety of pharmacological, antimicrobial activities of the live probiotic strain against several infectious agents involved in well-established acute and persistent watery diarrhoea and gastritis. Heat-treated cells and heat-stable secreted molecules trigger multiple strain-specific activities explaining the therapeutic efficacy of L. acidophilus LB. This review discusses the current body of knowledge on the antimicrobial mechanisms of action exerted by L. acidophilus LB demonstrated in in vitro and in vivo experimental studies, and the evidence for the therapeutic efficacy of this anti-infectious biotherapeutic agent proved in randomized clinical trials for the treatment of acute and persistent watery diarrhoea associated with several intestinal infectious diseases in humans. PMID:26770268

  19. 77 FR 9947 - Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry...

    Science.gov (United States)

    2012-02-21

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Early Clinical Trials With Live... availability of a document entitled ``Guidance for Industry: Early Clinical Trials With Live Biotherapeutic... submission of INDs for early clinical trials with live biotherapeutic products (LBPs). The guidance...

  20. Immunogenicity to Biotherapeutics – the role of Anti-drug Immune complexes

    Directory of Open Access Journals (Sweden)

    Murli eKrishna

    2016-02-01

    Full Text Available AbstractBiologic molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. Given the range of immune mediated adverse effects that have been documented to biologic drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biologic drugs in instances where there have been immune mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs elicited in vivo to a biotherapeutic; alongside with the structural, biophysical and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However many of the immune mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex intermediate (ICs that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing-ICs, risk factors that are either intrinsic to the therapeutic molecule or to the host which might predispose to IC mediated effects, and review the recent

  1. Immunogenicity to Biotherapeutics - The Role of Anti-drug Immune Complexes.

    Science.gov (United States)

    Krishna, Murli; Nadler, Steven G

    2016-01-01

    Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on

  2. Shedding light on biology of bacterial cells.

    Science.gov (United States)

    Schneider, Johannes P; Basler, Marek

    2016-11-01

    To understand basic principles of living organisms one has to know many different properties of all cellular components, their mutual interactions but also their amounts and spatial organization. Live-cell imaging is one possible approach to obtain such data. To get multiple snapshots of a cellular process, the imaging approach has to be gentle enough to not disrupt basic functions of the cell but also have high temporal and spatial resolution to detect and describe the changes. Light microscopy has become a method of choice and since its early development over 300 years ago revolutionized our understanding of living organisms. As most cellular components are indistinguishable from the rest of the cellular contents, the second revolution came from a discovery of specific labelling techniques, such as fusions to fluorescent proteins that allowed specific tracking of a component of interest. Currently, several different tags can be tracked independently and this allows us to simultaneously monitor the dynamics of several cellular components and from the correlation of their dynamics to infer their respective functions. It is, therefore, not surprising that live-cell fluorescence microscopy significantly advanced our understanding of basic cellular processes. Current cameras are fast enough to detect changes with millisecond time resolution and are sensitive enough to detect even a few photons per pixel. Together with constant improvement of properties of fluorescent tags, it is now possible to track single molecules in living cells over an extended period of time with a great temporal resolution. The parallel development of new illumination and detection techniques allowed breaking the diffraction barrier and thus further pushed the resolution limit of light microscopy. In this review, we would like to cover recent advances in live-cell imaging technology relevant to bacterial cells and provide a few examples of research that has been possible due to imaging

  3. Posttranslational Modifications and the Immunogenicity of Biotherapeutics

    Directory of Open Access Journals (Sweden)

    Roy Jefferis

    2016-01-01

    Full Text Available Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs, including quality control (QC in the endoplasmic reticulum (ER and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA; aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs, a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

  4. Mast cells: multitalented facilitators of protection against bacterial pathogens

    Science.gov (United States)

    Trivedi, Nikita H; Guentzel, M Neal; Rodriguez, Annette R; Yu, Jieh-Juen; Forsthuber, Thomas G; Arulanandam, Bernard P

    2014-01-01

    Mast cells are crucial effector cells evoking immune responses against bacterial pathogens. The positioning of mast cells at the host–environment interface, and the multitude of pathogen-recognition receptors and preformed mediator granules make these cells potentially the earliest to respond to an invading pathogen. In this review, the authors summarize the receptors used by mast cells to recognize invading bacteria and discuss the function of immune mediators released by mast cells in control of bacterial infection. The interaction of mast cells with other immune cells, including macrophages, dendritic cells and T cells, to induce protective immunity is highlighted. The authors also discuss mast cell-based vaccine strategies and the potential application in control of bacterial disease. PMID:23390944

  5. Bacterial Cell Surface Damage Due to Centrifugal Compaction

    NARCIS (Netherlands)

    Peterson, Brandon W.; Sharma, Prashant K.; van der Mei, Henny C.; Busscher, Henk J.

    2012-01-01

    Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we pr

  6. Osmotic Pressure, Bacterial Cell Walls, and Penicillin: A Demonstration.

    Science.gov (United States)

    Lennox, John E.

    1984-01-01

    An easily constructed apparatus that models the effect of penicillin on the structure of bacterial cells is described. Background information and procedures for using the apparatus during a classroom demonstration are included. (JN)

  7. Bacterial cell biology outside the streetlight.

    Science.gov (United States)

    Bulgheresi, Silvia

    2016-09-01

    As much as vertical transmission of microbial symbionts requires their deep integration into the host reproductive and developmental biology, symbiotic lifestyle might profoundly affect bacterial growth and proliferation. This review describes the reproductive oddities displayed by bacteria associated - more or less intimately - with multicellular eukaryotes.

  8. Bacterial cell biology outside the streetlight.

    Science.gov (United States)

    Bulgheresi, Silvia

    2016-09-01

    As much as vertical transmission of microbial symbionts requires their deep integration into the host reproductive and developmental biology, symbiotic lifestyle might profoundly affect bacterial growth and proliferation. This review describes the reproductive oddities displayed by bacteria associated - more or less intimately - with multicellular eukaryotes. PMID:27306428

  9. System-level design of bacterial cell cycle control

    OpenAIRE

    McAdams, Harley H.; Shapiro, Lucy

    2009-01-01

    Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. To a striking degree, the cell cycle regulation is a whole cell phenomenon. Phospho-signaling proteins and proteases dynamically deployed to specific loc...

  10. Beyond growth: novel functions for bacterial cell wall hydrolases.

    Science.gov (United States)

    Wyckoff, Timna J; Taylor, Jennifer A; Salama, Nina R

    2012-11-01

    The peptidoglycan cell wall maintains turgor pressure and cell shape of most bacteria. Cell wall hydrolases are essential, together with synthases, for growth and daughter cell separation. Recent work in diverse organisms has uncovered new cell wall hydrolases that act autonomously or on neighboring cells to modulate invasion of prey cells, cell shape, innate immune detection, intercellular communication, and competitor lysis. The hydrolases involved in these processes catalyze the cleavage of bonds throughout the sugar and peptide moities of peptidoglycan. Phenotypes associated with these diverse hydrolases reveal new functions of the bacterial cell wall beyond growth and division.

  11. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    Science.gov (United States)

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations.

  12. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    Science.gov (United States)

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations. PMID:9056304

  13. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  14. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  15. Mathematical Modeling of the Induced Mutation Process in Bacterial Cells

    Science.gov (United States)

    Belov, Oleg V.; Krasavin, Evgeny A.; Parkhomenko, Alexander Yu.

    2010-01-01

    A mathematical model of the ultraviolet (UV) irradiation-induced mutation process in bacterial cells Escherichia coli is developed. Using mathematical approaches, the whole chain of events is tracked from a cell exposure to the damaging factor to mutation formation in the DNA chain. An account of the key special features of the regulation of this genetic network allows predicting the effects induced by the cell exposure to certain UV energy fluence.

  16. The molecularly crowded cytoplasm of bacterialcCells : Dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells

    NARCIS (Netherlands)

    Trevors, J. T.; van Elsas, J. D.; Bej, A. K.

    2013-01-01

    In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but non-culturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell divi

  17. Heavy metal biosorption by bacterial cells

    Energy Technology Data Exchange (ETDEWEB)

    Vecchio, A.; Finoli, C.; Di Simine, D.; Andreoni, V. [Department of Food Science and Microbiology, State University, Milan (Italy)

    1998-06-01

    Microbial biomass provides available ligand groups on which metal ions bind by different mechanisms. Biosorption of these elements from aqueous solutions represents a remediation technology suitable for the treatment of metal-contaminated effluents. The purpose of the present investigation was the assessment of the capability of Brevibacterium sp. cells to remove bivalent ions, when present alone or in pairs, from aqueous solutions, using immobilized polyacrylamide cells of the microorganism in a flow-through system. The biosorption capacity of Brevibacterium cells was studied for lead, cadmium and copper. The metal cell binding capacity followed the order Cu > Pb > Cd, based on estimated q{sub max}. These values, expressed as mmol metal/g dry weight cells, were 0.54 for Cu, 0.36 for Pb and 0.14 for Cd. Polyacrylamide-gel immobilized cells were effective in Pb, Cu and Cd removal. Lead removal was not affected by the presence of Cd and Cu; lead instead inhibited Cd and Cu removal. The desorption of the metal, by fluxing a chelating solution, restored the metal binding capacity of the cells, thus affording the multiple use of the same biomass in the remediation treatment. (orig.) (orig.) With 5 figs., 4 tabs., 23 refs.

  18. Isolation of biologically active nanomaterial (inclusion bodies from bacterial cells

    Directory of Open Access Journals (Sweden)

    Peternel Špela

    2010-09-01

    Full Text Available Abstract Background In recent years bacterial inclusion bodies (IBs were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

  19. Expression of Bacterial β-Galactosidase in Animal Cells

    OpenAIRE

    An, Gynheung; Hidaka, Katsuhiko; Siminovitch, Louis

    1982-01-01

    A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.

  20. Spatial complexity and control of a bacterial cell cycle

    OpenAIRE

    Collier, Justine; Shapiro, Lucy

    2007-01-01

    A major breakthrough in understanding the bacterial cell cycle is the discovery that bacteria exhibit a high degree of intracellular organization. Chromosomal loci and many protein complexes are positioned at particular subcellular sites. In this review, we examine recently discovered control mechanisms that make use of dynamically localized protein complexes to orchestrate the Caulobacter crescentus cell cycle. Protein localization, notably of signal transduction proteins, chromosome partiti...

  1. A Bacterial Cell Shape-Determining Inhibitor.

    Science.gov (United States)

    Liu, Yanjie; Frirdich, Emilisa; Taylor, Jennifer A; Chan, Anson C K; Blair, Kris M; Vermeulen, Jenny; Ha, Reuben; Murphy, Michael E P; Salama, Nina R; Gaynor, Erin C; Tanner, Martin E

    2016-04-15

    Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials

  2. Fluid dynamics and noise in bacterial cell-cell and cell-surface scattering

    CERN Document Server

    Drescher, Knut; Cisneros, Luis H; Ganguly, Sujoy; Goldstein, Raymond E; 10.1073/pnas.1019079108

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell-cell and cell-surface scattering - the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report the first direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell-cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dom...

  3. Bounds on bacterial cell growth rates

    CERN Document Server

    Landy, Jonathan

    2013-01-01

    Recent experiments have shown that rod-like bacteria in nutrient-rich media grow in length at an exponential rate. Here, I point out that it is the elongated shape of these bacteria that allows for this behavior. Further, I show that when a bacterium's growth is limited by some nutrient -- taken in by the cell through a diffusion-to-capture process -- its growth is suppressed: In three-dimensional geometries, the length $L$ is bounded by $\\log L \\lesssim t^{1/2}$, while in two dimensions the length is bounded by a power-law form. Fits of experimental growth curves to these predicted, sub-exponential forms could allow for direct measures of quantities relating to cellular metabolic rates.

  4. 75 FR 63188 - Draft Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry...

    Science.gov (United States)

    2010-10-14

    ... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry: Early Clinical Trials With Live... availability of a draft document entitled ``Guidance for Industry: Early Clinical Trials with Live... submission of INDs for early clinical trials with live biotherapeutic products (LBPs). DATES: Although...

  5. Similar biotherapeutic products in Latin America. Regulation and opportunities for patients with autoimmune diseases

    Directory of Open Access Journals (Sweden)

    Desanvicente-Celis Z

    2013-01-01

    Full Text Available Zayrho Desanvicente-Celis, Julian Caro-Moreno, Mateo Enciso-Zuluaga, Juan-Manuel AnayaCenter for Autoimmune Diseases Research (CREA, Universidad del Rosario, Bogotá, ColombiaAbstract: Biotherapeutic products have revolutionized medicine, changing the way we can treat some chronic diseases, such as autoimmune diseases. The patent expiry and the high costs of reference biotherapeutic products, among other factors, have promoted interest in similar biotherapeutic products (SBPs, also known as biosimilars. The objective of developing an SBP is to manufacture a “highly similar” molecule to a reference biotherapeutic product, by conducting a comparability exercise that can demonstrate similar quality, safety, and efficacy. Regulations like those of the World Health Organization, the European Medicines Agency, and the Food and Drug Administration are international reference standards. Herein, we aim to point out the current status in Latin America on SBPs, focusing on regulatory issues within the context of autoimmune diseases. The regulations of Argentina, Peru, Chile, Guatemala, Panama and Costa Rica follow the World Health Organization guidelines. Other countries, such as Cuba, Mexico, Venezuela, and Brazil have regulations that take into account international standards combined with local features. In Colombia, a draft decree is under revision and the debate is ongoing. Some countries have already approved SBPs. Mexico, Chile, Ecuador, Bolivia, and Peru market SBPs of rituximab, and Colombia markets an SBP of etanercept. The advent of SBPs is definitely beneficial. Safety and efficacy must be ensured following clear and comprehensive regulations.Keywords: biological therapy, biotechnology, similar biotherapeutic product, autoimmune disease, Latin America

  6. The Role of Lipid Domains in Bacterial Cell Processes

    Directory of Open Access Journals (Sweden)

    Katarína Muchová

    2013-02-01

    Full Text Available Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

  7. Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

    Science.gov (United States)

    Byrne, W L; Murphy, C T; Cronin, M; Wirth, T; Tangney, M

    2014-12-28

    Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease. PMID:25466954

  8. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    Science.gov (United States)

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria. PMID:27088225

  9. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    Energy Technology Data Exchange (ETDEWEB)

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  10. Microarray Analysis to Monitor Bacterial Cell Wall Homeostasis.

    Science.gov (United States)

    Hong, Hee-Jeon; Hesketh, Andy

    2016-01-01

    Transcriptomics, the genome-wide analysis of gene transcription, has become an important tool for characterizing and understanding the signal transduction networks operating in bacteria. Here we describe a protocol for quantifying and interpreting changes in the transcriptome of Streptomyces coelicolor that take place in response to treatment with three antibiotics active against different stages of peptidoglycan biosynthesis. The results defined the transcriptional responses associated with cell envelope homeostasis including a generalized response to all three antibiotics involving activation of transcription of the cell envelope stress sigma factor σ(E), together with elements of the stringent response, and of the heat, osmotic, and oxidative stress regulons. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. The principles behind the protocol are transferable to the study of cell envelope homeostatic mechanisms probed using alternative chemical/environmental insults or in other bacterial strains. PMID:27311662

  11. A cell cycle and nutritional checkpoint controlling bacterial surface adhesion.

    Directory of Open Access Journals (Sweden)

    Aretha Fiebig

    2014-01-01

    Full Text Available In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ. Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.

  12. Lung Dendritic Cells Facilitate Extrapulmonary Bacterial Dissemination during Pneumococcal Pneumonia

    Directory of Open Access Journals (Sweden)

    Alva eRosendahl

    2013-06-01

    Full Text Available Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DC-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DC-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9 in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection.

  13. Prodigiosin inhibits motility and activates bacterial cell death revealing molecular biomarkers of programmed cell death.

    Science.gov (United States)

    Darshan, N; Manonmani, H K

    2016-12-01

    The antimicrobial activity of prodigiosin from Serratia nematodiphila darsh1, a bacterial pigment was tested against few food borne bacterial pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The mode of action of prodigiosin was studied. Prodigiosin induced bactericidal activity indicating a stereotypical set of biochemical and morphological feature of Programmed cell death (PCD). PCD involves DNA fragmentation, generation of ROS, and expression of a protein with caspase-like substrate specificity in bacterial cells. Prodigiosin was observed to be internalized into bacterial cells and was localized predominantly in the membrane and the nuclear fraction, thus, facilitating intracellular trafficking and then binding of prodigiosin to the bacterial DNA. Corresponding to an increasing concentration of prodigiosin, the level of certain proteases were observed to increase in bacteria studied, thus initiating the onset of PCD. Prodigiosin at a sub-inhibitory concentration inhibits motility of pathogens. Our observations indicated that prodigiosin could be a promising antibacterial agent and could be used in the prevention of bacterial infections. PMID:27460563

  14. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    Science.gov (United States)

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  15. Softness of the bacterial cell wall of Streptococcus mitis as probed by micro-electrophoresis

    NARCIS (Netherlands)

    Vadillo-Rodriguez, V.; Busscher, H.J.; Norde, W.; Mei, van der H.C.

    2002-01-01

    Chemical and structural complexity of bacterial cell surfaces complicate accurate quantification of cell surfaces properties. The presence of fibrils, fimbriae or other surface appendages on bacterial cell surfaces largely influence those properties and would therefore play a major function in inter

  16. Transmission electron microscopy and atomic force microscopy characterization of nickel deposition on bacterial cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Recently bacterial cells have become attractive biological templates for the fabrication of metal nano- structures or nanomaterials due to their inherent small size, various standard geometrical shapes and abundant source. In this paper, nickel-coated bacterial cells (gram-negative bacteria of Escherichia coli) were fabricated via electroless chemical plating. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) characterization results reveal evident morphological difference between bacterial cells before and after deposition with nickel. The bare cells with smooth surface presented transverse outspreading effect at mica surface. Great changes took place in surface roughness for those bacterial cells after metallization. A large number of nickel nanoparticles were observed to be equably distributed at bacterial surface after activation and subsequent metallization. Furthermore, ultra thin section analytic results validated the presence and uniformity of thin nickel coating at bacterial surface after metallization.

  17. Principles of bacterial cell-size determination revealed by cell wall synthesis perturbations

    OpenAIRE

    Carolina Tropini; Timothy K. Lee; Jen Hsin; Samantha M. Desmarais; Tristan Ursell; Russell D. Monds; Kerwyn Casey Huang

    2014-01-01

    Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cyto...

  18. (p)ppGpp and the bacterial cell cycle

    Indian Academy of Sciences (India)

    Aanisa Nazir; Rajendran Harinarayanan

    2016-06-01

    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  19. (p)ppGpp and the bacterial cell cycle.

    Science.gov (United States)

    Nazir, Aanisa; Harinarayanan, Rajendran

    2016-06-01

    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  20. ASSESSMENT OF BIOTHERAPEUTIC POTENTIAL OF PIMENTA DIOICA (ALLSPICE LEAF EXTRACT

    Directory of Open Access Journals (Sweden)

    Dr Pratima Khandelwal et al

    2012-09-01

    Full Text Available All-spice (pimenta is one of the under-utilized resources available in the tropical regions of the globe. It is a variety of sweet pepper used as a spice and its leaves are used for traditional culinary purpose. Researchers have studied the antioxidant potentials of the berries of the plant, but no documented work is reported on its stem, leaf and roots for antimicrobial properties. Thus, the present investigation was carried out to access the antimicrobial and anti-oxidation potentials of leaf extracts using three solvent systems, (Aqueous, acetone and methanol. All solvent systems at different concentrations were evaluated for antibacterial, antifungal and reducing capacity against selected bacterial and fungal pathogens; zone of inhibition was exhibited by methanol leaf extracts in decreasing order for Escherichia coli, Bacillus cereus, Salmonella typhimurium, and Staphylococcus aureus. Lesser inhibitory zones were obtained by acetone leaf extracts, whereas, Klebsiella pneumonia and Pseudomonas aeruginosa were not inhibited by any extracts. Aqueous extract demonstrated no inhibitory activity against tested bacterial pathogens. All the three leaf extracts were found to be ineffective against fungal strains (Fusarium oxysporum, Aspergillus niger, Aspergillus flavus and Candida albicans tested. Protein content in each extract was determined and reducing capability was estimated which was found to be high in methanol and acetone extract whereas aqueous extract showed low reducing ability.

  1. Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.

    Directory of Open Access Journals (Sweden)

    Sandra L Diaz

    Full Text Available BACKGROUND: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc, but can metabolically incorporate it from dietary sources (particularly red meat and milk into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA, Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated

  2. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    of contact. Staphylococcus xylosus DSM 20266 and Staphylococcus epidermidis DSM 20044 showed much higher adhesion forces than Pseudomonas fluorescens AH1, but bond strengthening by P. aeruginosa (2 s) was faster than for the staphylococci (10 s) . Escherichia coli DSM 429, which was the only strain unable...... to form biofilm, showed almost no adhesion to any surface. The differences between staphylococci and P. fluorescens in adhesion pattern reflects their differences in the composition of extracellular adhesins. Both adhesion force and rupture length were significantly smaller on mica compared to glass....... Staphylococci adhere stronger on fresh glass than on hydrophilic glass, while the weaker adhesion by P. fluorescens was similar on both types of glass. These results confirmed the importance of surface hydrophobicity in bacterial adhesion. This study has demonstrated that single-cell force spectroscopy allows...

  3. Bacterial protein toxins : tools to study mammalian molecular cell biology

    NARCIS (Netherlands)

    Wüthrich, I.W.

    2014-01-01

    Bacterial protein toxins are genetically encoded proteinaceous macromolecules that upon exposure causes perturbation of cellular metabolism in a susceptible host. A bacterial toxin can work at a distance from the site of infection, and has direct and quantifiable actions. Bacterial protein toxins ca

  4. Bacterial Cell Wall-Induced Arthritis: Chemical Composition and Tissue Distribution of Four Lactobacillus Strains

    OpenAIRE

    Šimelyte, Egle; Rimpiläinen, Marja; Lehtonen, Leena; Zhang, Xiang; Toivanen, Paavo

    2000-01-01

    To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentum did not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentum was different. Likewise, all arthritogenic cell walls...

  5. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W;

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients...

  6. Global Foot-and-Mouth Disease Research Update and Gap Analysis: 5 - Biotherapeutics and Disinfectants.

    Science.gov (United States)

    Robinson, L; Knight-Jones, T J D; Charleston, B; Rodriguez, L L; Gay, C G; Sumption, K J; Vosloo, W

    2016-06-01

    We assessed knowledge gaps in foot-and-mouth disease (FMD) research. Findings are reported in a series of papers, and in this article, we consider biotherapeutics and disinfectants. The study took the form of a literature review (2011-2015) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. While vaccines will remain the key immunological intervention used against FMD virus (FMDV) for the foreseeable future, it takes a few days for the immune system to respond to vaccination. In an outbreak situation, protection could potentially be provided during this period by the application of rapid, short-acting biotherapeutics, aiming either to stimulate a non-specific antiviral state in the animal or to specifically inhibit a part of the viral life cycle. Certain antiviral cytokines have been shown to promote rapid protection against FMD; however, the effects of different immune-modulators appear to vary across species in ways and for reasons that are not yet understood. Major barriers to the effective incorporation of biotherapeutics into control strategies are cost, limited understanding of their effect on subsequent immune responses to vaccines and uncertainty about their potential impact if used for disease containment. Recent research has highlighted the importance of environmental contamination in FMDV transmission. Effective disinfectants for FMDV have long been available, but research is being conducted to further develop methods for quantitatively evaluating their performance under field, or near-field, conditions. During outbreaks in South Korea in 2010 there was public concern about potential environmental contamination after the mass use of disinfectant and mass burial of culled stock; this should be considered during outbreak contingency planning. PMID:27320166

  7. DCVax®-L—Developed by Northwest Biotherapeutics

    OpenAIRE

    Polyzoidis, Stavros; Ashkan, Keyoumars

    2014-01-01

    Dendritic cell (DC) immunotherapy is emerging as a potential addition to the standard of care in the treatment of glioblastoma multiforme (GBM). In the last decade or so various research groups have conducted phase I and II trials of DC-immunotherapy on patients with newly diagnosed (ND) and recurrent GBM and other high-grade gliomas in an attempt to improve the poor prognosis. Results show an increase in overall survival (OS), while vaccination-related side effects are invariably mild. North...

  8. Guidelines for monitoring bulk tank milk somatic cell and bacterial counts.

    Science.gov (United States)

    Jayarao, B M; Pillai, S R; Sawant, A A; Wolfgang, D R; Hegde, N V

    2004-10-01

    This study was conducted to establish guidelines for monitoring bulk tank milk somatic cell count and bacterial counts, and to understand the relationship between different bacterial groups that occur in bulk tank milk. One hundred twenty-six dairy farms in 14 counties of Pennsylvania participated, each providing one bulk tank milk sample every 15 d for 2 mo. The 4 bulk tank milk samples from each farm were examined for bulk tank somatic cell count and bacterial counts including standard plate count, preliminary incubation count, laboratory pasteurization count, coagulase-negative staphylococcal count, environmental streptococcal count, coliform count, and gram-negative noncoliform count. The milk samples were also examined for presence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The bacterial counts of 4 bulk tank milk samples examined over an 8-wk period were averaged and expressed as mean bacterial count per milliliter. The study revealed that an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae was significantly associated with an increased bulk tank somatic cell count. Paired correlation analysis showed that there was low correlation between different bacterial counts. Bulk tank milk with low (standard plate count also had a significantly low level of mean bulk tank somatic cell count (count (count (counts (count (count was less likely to be associated with somatic cell or other bacterial counts. Herd size and farm management practices had considerable influence on somatic cell and bacterial counts in bulk tank milk. Dairy herds that used automatic milking detachers, sand as bedding material, dip cups for teat dipping instead of spraying, and practiced pre-and postdipping had significantly lower bulk tank somatic cell and/or bacterial counts. In conclusion, categorized bulk tank somatic cell and bacterial counts could serve as indicators and facilitate monitoring of herd udder health and milk

  9. CLA-1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells

    OpenAIRE

    Vishnyakova, Tatyana G.; Kurlander, Roger; Bocharov, Alexander V.; Baranova, Irina N.; CHEN, ZHIGANG; Abu-Asab, Mones S.; Tsokos, Maria; Malide, Daniela; Basso, Federica; Remaley, Alan; Csako, Gyorgy; Eggerman, Thomas L.; Patterson, Amy P.

    2006-01-01

    CD36 and LIMPII analog 1, CLA-1, and its splicing variant, CLA-2 (SR-BI and SR-BII in rodents), are human high density lipoprotein receptors with an identical extracellular domain which binds a spectrum of ligands including bacterial cell wall components. In this study, CLA-1- and CLA-2-stably transfected HeLa and HEK293 cells demonstrated several-fold increases in the uptake of various bacteria over mock-transfected cells. All bacteria tested, including both Gram-negatives (Escherichia coli ...

  10. Bacterial colonization of host cells in the absence of cholesterol.

    Directory of Open Access Journals (Sweden)

    Stacey D Gilk

    2013-01-01

    Full Text Available Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/- mouse embryonic fibroblasts (MEFs. DHCR24(-/- MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/- MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/- MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(Vβ(3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/- MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

  11. Cholesterol-Lowering Probiotics as Potential Biotherapeutics for Metabolic Diseases

    Directory of Open Access Journals (Sweden)

    Manoj Kumar

    2012-01-01

    Full Text Available Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface

  12. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  13. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  14. STD NMR spectroscopy: a case study of fosfomycin binding interactions in living bacterial cells

    Energy Technology Data Exchange (ETDEWEB)

    Milagre, Cintia D.F.; Cabeca, Luis Fernando; Martins, Lucas G.; Marsaioli, Anita J., E-mail: anita@iq [Universidade Estadual de Campinas (IQ/UNICAMP), SP (Brazil). Inst. de Quimica

    2011-07-01

    A saturation transfer difference (STD) NMR experiment was successfully employed to observe the binding interactions of fosfomycin resistant and non-resistant bacterial strains using living cell suspensions, without the need for isotopic labelling of the ligand or receptor. (author)

  15. Phase Diagram of Collective Motion of Bacterial Cells in a Shallow Circular Pool

    CERN Document Server

    Wakita, Jun-ichi; Yamamoto, Ken; Katori, Makoto; Yamada, Yasuyuki

    2015-01-01

    The collective motion of bacterial cells in a shallow circular pool is systematically studied using the bacterial species $Bacillus$ $subtilis$. The ratio of cell length to pool diameter (i.e., the reduced cell length) ranges from 0.06 to 0.43 in our experiments. Bacterial cells in a circular pool show various types of collective motion depending on the cell density in the pool and the reduced cell length. The motion is classified into six types, which we call random motion, turbulent motion, one-way rotational motion, two-way rotational motion, random oscillatory motion, and ordered oscillatory motion. Two critical values of reduced cell lengths are evaluated, at which drastic changes in collective motion are induced. A phase diagram is proposed in which the six phases are arranged.

  16. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency

    OpenAIRE

    Qiang Tu; Jia Yin; Jun Fu; Jennifer Herrmann; Yuezhong Li; Yulong Yin; Francis Stewart, A.; Rolf Müller; Youming Zhang

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and re...

  17. Bacterial toxins fuel disease progression in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise Maria;

    2013-01-01

    . Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL. Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel......In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus...

  18. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@thep-center.org [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2014-05-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  19. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S;

    2001-01-01

    (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency......Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  20. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard;

    2010-01-01

    that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular...

  1. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    S Kaur

    2012-01-01

    Full Text Available Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P0.05 in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

  2. Cell order in bacterial swarms arises from reversals of moving direction

    Science.gov (United States)

    Wu, Yilin; Jiang, Yi; Kaiser, Dale; Alber, Mark

    2010-03-01

    Bacterial swarms are a beautiful example of the emergent behavior of systems of self-propelled rods. In swarming rod-shaped bacteria cells move smoothly even though they are packed together in high density. Experimental evidence shows that long-distance signaling is not required for bacterial swarming. It naturally raises the question how a swarm develops its order. Using a biomechanical model, we show here that regular periodic reversals of gliding direction in general systems of self-propelled rod shaped bacteria can lead to the extensive ordering of cells. We also show that an optimal reversal period and an optimal cell shape exist for producing such order. Given the observations of reversing behavior in several bacterial species,we suggest that the capacity to swarm depends less on the motility engine employed by individual cells, but more on the behavioral algorithm that enhances the flow of densely packed cells near the swarming edge.

  3. Molecular Architecture of the Bacterial Flagellar Motor in Cells

    OpenAIRE

    Zhao, Xiaowei; Norris, Steven J; Liu, Jun

    2014-01-01

    The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and ...

  4. TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens

    OpenAIRE

    Peyssonnaux, Carole; Zinkernagel, Annelies S.; Datta, Vivekanand; Lauth, Xavier; Johnson, Randall S; Nizet, Victor

    2006-01-01

    Hepcidin is an antimicrobial peptide secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. Here we demonstrate the endogenous expression of hepcidin by macrophages and neutrophils in vitro and in vivo. These myeloid cell types produced hepcidin in response to bacterial pathogens in a toll-like receptor 4 (TLR4)-dependent fashion. Conversely, bacterial stimulation of macrophages triggered a TLR4-dependent reduction in the iron exporter ferroportin. ...

  5. Single-molecule investigations of the stringent response machinery in living bacterial cells

    OpenAIRE

    English, Brian P.; Hauryliuk, Vasili; Sanamrad, Arash; Tankov, Stoyan; Dekker, Nynke H.; Elf, Johan

    2011-01-01

    The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that Re...

  6. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  7. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  8. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. PMID:24681053

  9. Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

    Directory of Open Access Journals (Sweden)

    Stella E Autenrieth

    2012-02-01

    Full Text Available Dendritic cells (DCs as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye. We used CD11c-diphtheria toxin (DT mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

  10. Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

    Science.gov (United States)

    Sharkey, Michael A; Chebbi, Ahmed; McDonnell, Kevin A; Staunton, Claire; Dowling, Denis P

    2015-01-01

    The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

  11. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level

    Science.gov (United States)

    Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A.; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

    2015-01-01

    Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake. PMID:26656111

  12. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level.

    Science.gov (United States)

    Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

    2015-12-11

    Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.

  13. Mechanisms of bacterial morphogenesis: evolutionary cell biology approaches provide new insights.

    Science.gov (United States)

    Jiang, Chao; Caccamo, Paul D; Brun, Yves V

    2015-04-01

    How Darwin's "endless forms most beautiful" have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating "evolutionary thinking" into bacterial cell biology in the genomic era.

  14. Search for MicroRNAs Expressed by Intracellular Bacterial Pathogens in Infected Mammalian Cells

    Science.gov (United States)

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A.; Xet-Mull, Ana M.; Sisk, Dana M.; Smith, Kristen L. Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H.; Tobin, David M.; Cullen, Bryan R.

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin. PMID:25184567

  15. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Directory of Open Access Journals (Sweden)

    Yuki Furuse

    Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  16. Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

    Science.gov (United States)

    Bucher, Tabitha; Oppenheimer-Shaanan, Yaara; Savidor, Alon; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

    2015-12-01

    In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment. PMID:26472159

  17. Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

    Science.gov (United States)

    Bucher, Tabitha; Oppenheimer-Shaanan, Yaara; Savidor, Alon; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

    2015-12-01

    In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.

  18. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  19. Solving the mysteries of the bacterial cell – application of novel techniques in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Magdalena Donczew

    2011-01-01

    Full Text Available We have reviewed how the development of fluorescent markers, triggered by the discovery of green fluorescence protein and its other color variants leading to the establishment of methods for studies of protein interactions with application of fluorescent proteins, affected the view of bacterial cell organization. Application of the new microscopic methods allowed localization of proteins and chromosomal regions, and observation of their migration in real time. These studies revealed the spatial organization of bacterial cells which includes specific subcellular localization of proteins, the presence of dynamic cytoskeletal structures, orchestrated and active segregation of chromosomes, and spatiotemporal gene regulation.

  20. Bacterial swarmer cells in confinement: A mesoscale hydrodynamic simulation study

    CERN Document Server

    Eisenstecken, Thomas; Winkler, Roland G

    2016-01-01

    A wide spectrum of Peritrichous bacteria undergo considerable physiological changes when they are inoculated onto nutrition-rich surfaces and exhibit a rapid and collective migration denoted as swarming. Thereby, the length of such swarmer cells and their number of flagella increases substantially. In this article, we investigated the properties of individual E. coli-type swarmer cells confined between two parallel walls via mesoscale hydrodynamic simulations, combining molecular dynamics simulations of the swarmer cell with the multiparticle particle collision dynamics approach for the embedding fluid. E. coli-type swarmer cells are three-times longer than their planktonic counter parts, but their flagella density is comparable. By varying the wall separation, we analyze the confinement effect on the flagella arrangement, on the distribution of cells in the gap between the walls, and on the cell dynamics. We find only a weak dependence of confinement on the bundle structure and dynamics. The distribution of ...

  1. On the chronology and topography of bacterial cell division.

    Science.gov (United States)

    Vicente, M; Palacios, P; Dopazo, A; Garrido, T; Pla, J; Aldea, M

    1991-01-01

    Gene products that play a role in the formation of cell septum should be expected to be endowed with a set of specific properties. In principle, septal proteins should be located at the cell envelope. The expression of division genes should ensure the synthesis of septal proteins at levels commensurate with the needs of cell division at different rates of cell duplication. We have results indicating that some fts genes located within the 2.5-min cluster in the Escherichia coli chromosome conform to these predictions.

  2. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater

    Science.gov (United States)

    Becker, M.W.; Collins, S.A.; Metge, D.W.; Harvey, R.W.; Shapiro, A.M.

    2004-01-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates. ?? 2003 Elsevier B.V. All rights reserved.

  3. Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation.

    Science.gov (United States)

    Boix-Amorós, Alba; Collado, Maria C; Mira, Alex

    2016-01-01

    Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system.

  4. Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation.

    Science.gov (United States)

    Boix-Amorós, Alba; Collado, Maria C; Mira, Alex

    2016-01-01

    Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system. PMID:27148183

  5. Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

    Directory of Open Access Journals (Sweden)

    Andrew S Brown

    2016-06-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC, which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

  6. A mechanistic stochastic framework for regulating bacterial cell division.

    Science.gov (United States)

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A; Singh, Abhyudai

    2016-01-01

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size. PMID:27456660

  7. A mechanistic stochastic framework for regulating bacterial cell division.

    Science.gov (United States)

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A; Singh, Abhyudai

    2016-07-26

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size.

  8. Development of Phage Lysin LysA2 for Use in Improved Purity Assays for Live Biotherapeutic Products.

    Science.gov (United States)

    Dreher-Lesnick, Sheila M; Schreier, Jeremy E; Stibitz, Scott

    2015-12-01

    Live biotherapeutic products (LBPs), commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, but there is also growing interest in evaluating such products for treatment of specific diseases. While over-the-counter probiotics are generally viewed as very safe, at least in healthy individuals, it must be remembered that clinical studies to assess these products may be done in individuals whose defenses are compromised, such as through a disease process, immunosuppressive clinical treatment, or an immature or aging immune system. One of the major safety criteria for LBPs used in clinical studies is microbial purity, i.e., the absence of extraneous, undesirable microorganisms. The main goal of this project is to develop recombinant phage lysins as reagents for improved purity assays for LBPs. Phage lysins are hydrolytic enzymes containing a cell binding domain that provides specificity and a catalytic domain responsible for lysis and killing. Our approach is to use recombinant phage lysins to selectively kill target product bacteria, which when used for purity assays will allow for outgrowth of potential contaminants under non-selective conditions, thus allowing an unbiased assessment of the presence of contaminants. To develop our approach, we used LysA2, a phage lysin with reported activity against a broad range of Lactobacillus species. We report the lytic profile of a non-tagged recombinant LysA2 against Lactobacillus strains in our collection. We also present a proof-of-concept experiment, showing that addition of partially purified LysA2 to a culture of Lactobacillus jensenii (L. jensenii) spiked with low numbers of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus ) effectively eliminates or knocks down L

  9. Enhanced metalloadsorption of bacterial cells displaying poly-His peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, C.; Cebolla, A.; Lorenzo, V. de [CSIC, Madrid (Spain)

    1996-08-01

    The properties of Escherichia coli cells, acquired by cell surface presentation of one or two hexahistidine (His) clusters carried by the outer membrane LamB protein, have been examined. Strains producing LamB hybrids with the His chains accumulated greater than 11-fold more Cd{sup 2} than E. coli cells expressing the protein without the His insert. Furthermore, the hexa-His chains on the cell surface caused cells to adhere reversibly to a Ni{sup 2+}-containing solid matrix in a metal-dependent fashion. Thus, expression of poly-His peptides enables bacteria to act as a metalloaffinity adsorbent. These results open up the possibility for biosorption of heavy ions using engineered microorganisms. 32 refs., 3 figs.

  10. Water Diffusion from a Bacterial Cell in Low-Moisture Foods.

    Science.gov (United States)

    Syamaladevi, Roopesh M; Tang, Juming; Zhong, QingPing

    2016-09-01

    We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low-moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze-dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10(-12) and 10(-10) m(2) /s for bacterial cells, the predicted equilibration times (teq ) ranged from 8.24×10(-4) to 8.24×10(-2) s. Considering a cell membrane barrier with a lower water diffusion coefficient (10(-15) m(2) /s) around the bacterial cell with a water diffusion coefficient of 10(-12) m(2) /s, the teq predicted using COMSOL Multiphysics program was 3.8×10(-1) s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature. PMID:27505687

  11. Water Diffusion from a Bacterial Cell in Low-Moisture Foods.

    Science.gov (United States)

    Syamaladevi, Roopesh M; Tang, Juming; Zhong, QingPing

    2016-09-01

    We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low-moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze-dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10(-12) and 10(-10) m(2) /s for bacterial cells, the predicted equilibration times (teq ) ranged from 8.24×10(-4) to 8.24×10(-2) s. Considering a cell membrane barrier with a lower water diffusion coefficient (10(-15) m(2) /s) around the bacterial cell with a water diffusion coefficient of 10(-12) m(2) /s, the teq predicted using COMSOL Multiphysics program was 3.8×10(-1) s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature.

  12. Nanoscale Electric Permittivity of Single Bacterial Cells at Gigahertz Frequencies by Scanning Microwave Microscopy.

    Science.gov (United States)

    Biagi, Maria Chiara; Fabregas, Rene; Gramse, Georg; Van Der Hofstadt, Marc; Juárez, Antonio; Kienberger, Ferry; Fumagalli, Laura; Gomila, Gabriel

    2016-01-26

    We quantified the electric permittivity of single bacterial cells at microwave frequencies and nanoscale spatial resolution by means of near-field scanning microwave microscopy. To this end, calibrated complex admittance images have been obtained at ∼19 GHz and analyzed with a methodology that removes the nonlocal topographic cross-talk contributions and thus provides quantifiable intrinsic dielectric images of the bacterial cells. Results for single Escherichia coli cells provide a relative electric permittivity of ∼4 in dry conditions and ∼20 in humid conditions, with no significant loss contributions. Present findings, together with the ability of microwaves to penetrate the cell membrane, open an important avenue in the microwave label-free imaging of single cells with nanoscale spatial resolution.

  13. Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals.

    Science.gov (United States)

    Holtz, Barry R; Berquist, Brian R; Bennett, Lindsay D; Kommineni, Vally J M; Munigunti, Ranjith K; White, Earl L; Wilkerson, Don C; Wong, Kah-Yat I; Ly, Lan H; Marcel, Sylvain

    2015-10-01

    Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever. PMID:26387511

  14. Lubricin: a novel potential biotherapeutic approaches for the treatment of osteoarthritis.

    Science.gov (United States)

    Bao, Jia-Peng; Chen, Wei-Ping; Wu, Li-Dong

    2011-06-01

    Osteoarthritis (OA) is a multi-factor disorder of sinovial joints, which characterized by escalated degeneration and loss of articular cartilage. Treatment of OA is a critical unmet need in medicine for regeneration of damaged articular cartilage in elderly. On the other hand, lubricin, a glycoprotein specifically synthesized by chondrocytes located at the surface of articular cartilage, has been shown to provide boundary lubrication of congruent articular surfaces under conditions of high contact pressure and near zero sliding speed. Lubrication of these surfaces is critical to normal joint function, while different gene expressions of lubricin had been found in the synovium of rheumatoid arthritis (RA) and OA. Moreover, mutations or lacking of lubricin gene have been shown to link to the joint disease such as camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), synovial hyperplasia and failure of joint function, suggesting an important role of lubricin in the pathogenesis of these joint disease. Recent studies demonstrate that administration with recombinant lubricin in the joint cavity would be effective in the prevention of cartilage degeneration in animal OA models. Therefore, a treatment with lubricin which would protect cartilage in vivo would be desirable. This article reviews recent findings with regard to the possible role of lubricin in the progression of OA, and further discusses lubricin as a novel potential biotherapeutic approaches for the treatment of OA.

  15. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

    Science.gov (United States)

    Riba, J; Gleichmann, T; Zimmermann, S; Zengerle, R; Koltay, P

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  16. Heterotrophic free-living and particle-bound bacterial cell size in the river Cauvery and its downstream tributaries

    Indian Academy of Sciences (India)

    T S Harsha; Sadanand M Yamakanamardi; M Mahadevaswamy

    2007-03-01

    This is the first comprehensive study on planktonic heterotrophic bacterial cell size in the river Cauvery and its important tributaries in Karnataka State, India. The initial hypothesis that the mean cell size of planktonic heterotrophic bacteria in the four tributaries are markedly different from each other and also from that in the main river Cauvery was rejected, because all five watercourses showed similar planktonic heterotrophic bacterial cell size. Examination of the correlation between mean heterotrophic bacterial cell size and environmental variables showed four correlations in the river Arkavathy and two in the river Shimsha. Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial cell size was due to biological oxygen demand (BOD) in the river Arkavathy, 11% due to surface water velocity (SWV) in the river Cauvery and 11% due to temperature in the river Kapila. Heterotrophic particle-bound bacterial cell size variation was 28% due to chloride and BOD in the river Arkavathy, 11% due to conductivity in the river Kapila and 8% due to calcium in the river Cauvery. This type of relationship between heterotrophic bacterial cell size and environmental variables suggests that, though the mean heterotrophic bacterial cell size was similar in all the five water courses, different sets of environmental variables apparently control the heterotrophic bacterial cell size in the various water bodies studied in this investigation. The possible cause for this environmental (bottom–up) control is discussed.

  17. Synchronization of Caulobacter crescentus for investigation of the bacterial cell cycle.

    Science.gov (United States)

    Schrader, Jared M; Shapiro, Lucy

    2015-04-08

    The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

  18. Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

    Science.gov (United States)

    Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

    2016-12-01

    We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. PMID:27554145

  19. Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

    Science.gov (United States)

    Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

    2016-12-01

    We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process.

  20. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  1. Homeostatic interplay between bacterial cell-cell signaling and iron in virulence.

    Directory of Open Access Journals (Sweden)

    Ronen Hazan

    2010-03-01

    Full Text Available Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.

  2. Lactic acid bacterial cell factories for gamma-aminobutyric acid.

    Science.gov (United States)

    Li, Haixing; Cao, Yusheng

    2010-11-01

    Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.

  3. The Role of Lipid Domains in Bacterial Cell Processes

    OpenAIRE

    Katarína Muchová; Imrich Barák

    2013-01-01

    Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins....

  4. Effects of bacterial cells and two types of extracellular polymers on bioclogging of sand columns

    Science.gov (United States)

    Xia, Lu; Zheng, Xilai; Shao, Haibing; Xin, Jia; Sun, Zhaoyue; Wang, Leyun

    2016-04-01

    Microbially induced reductions in the saturated hydraulic conductivity, Ks, of natural porous media, conventionally called bioclogging, occurs frequently in natural and engineered subsurface systems. Bioclogging can affect artificial groundwater recharge, in situ bioremediation of contaminated aquifers, or permeable reactive barriers. In this study, we designed a series of percolation experiments to simulate the growth and metabolism of bacteria in sand columns. The experimental results showed that the bacterial cell amount gradually increased to a maximum of 8.91 log10 CFU/g sand at 144 h during the bioclogging process, followed by a decrease to 7.89 log10 CFU/g sand until 336 h. The same variation pattern was found for the concentration of tightly bound extracellular polymeric substances (TB-EPS), which had a peak value of 220.76 μg/g sand at 144 h. In the same experiments, the concentration of loosely bound extracellular polymeric substances (LB-EPS) increased sharply from 54.45 to 575.57 μg/g sand in 192 h, followed by a slight decline to 505.04 μg/g sand. The increase of the bacterial cell amount along with the other two concentrations could reduce the Ks of porous media, but their relative contributions varied to a large degree during different percolation stages. At the beginning of the tests (e.g., 48 h before), bacterial cells were likely responsible for the Ks reduction of porous media because no increase was found for the other two concentrations. With the accumulation of cells and EPS production from 48 to 144 h, both were important for the reduction of Ks. However, in the late period of percolation tests from 144 to 192 h, LB-EPS was probably responsible for the further reduction of Ks, as the bacterial cell amount and TB-EPS concentration decreased. Quantitative contributions of bacterial cell amount and the two types of extracellular polymers to Ks reductions were also evaluated.

  5. Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy.

    Science.gov (United States)

    Volle, C B; Ferguson, M A; Aidala, K E; Spain, E M; Núñez, M E

    2008-11-15

    Bacterial biofilms were imaged by atomic force microscopy (AFM), and their elasticity and adhesion to the AFM tip were determined from a series of tip extension and retraction cycles. Though the five bacterial strains studied included both Gram-negative and -positive bacteria and both environmental and laboratory strains, all formed simple biofilms on glass surfaces. Cellular spring constants, determined from the extension portion of the force cycle, varied between 0.16+/-0.01 and 0.41+/-0.01 N/m, where larger spring constants were measured for Gram-positive cells than for Gram-negative cells. The nonlinear regime in the extension curve depended upon the biomolecules on the cell surface: the extension curves for the smooth Gram-negative bacterial strains with the longest lipopolysaccharides on their surface had a larger nonlinear region than the rough bacterial strain with shorter lipopolysaccharides on the surface. Adhesive forces between the retracting silicon nitride tip and the cells varied between cell types in terms of the force components, the distance components, and the number of adhesion events. The Gram-negative cells' adhesion to the tip showed the longest distance components, sometimes more than 1 microm, whereas the shortest distance adhesion events were measured between the two Gram-positive cell types and the tip. Fixation of free-swimming planktonic cells by NHS and EDC perturbed both the elasticity and the adhesive properties of the cells. Here we consider the biochemical meaning of the measured physical properties of simple biofilms and implications to the colonization of surfaces in the first stages of biofilm formation. PMID:18815013

  6. Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies

    NARCIS (Netherlands)

    van Dam, V.; Olrichs, N.K.; Breukink, E.J.

    2009-01-01

    Wall chart: The predominant component of the bacterial cell wall, peptidoglycan, consists of long alternating stretches of aminosugar subunits interlinked in a large three-dimensional network and is formed from precursors through several cytosolic and membrane-bound steps. The high tolerance of the

  7. Disinfection byproduct formation from chlorination of pure bacterial cells and pipeline biofilms.

    Science.gov (United States)

    Wang, Jun-Jian; Liu, Xin; Ng, Tsz Wai; Xiao, Jie-Wen; Chow, Alex T; Wong, Po Keung

    2013-05-15

    Disinfection byproduct (DBP) formation is commonly attributed to the reaction between natural organic matters and disinfectants, yet few have considered the contribution from disinfecting bacterial materials - the essential process of water disinfection. Here, we explored the DBP formation from chlorination and chloramination of Escherichia coli and found that most selected DBPs were detectable, including trihalomethanes, haloacetonitriles, chloral hydrate, chloropicrin, and 1,1,1-trichloro-2-propanone. A positive correlation (P = 0.08-0.09) between DBP formation and the log reduction of E. coli implied that breaking down of bacterial cells released precursors for DBP formation. As Pseudomonas aeruginosa is a dominant bacterial species in pipeline biofilms, the DBP formation potentials (DBPFPs) from its planktonic cells and biofilms were characterized. Planktonic cells formed 7-11 times greater trihalomethanes per carbon of those from biofilms but significantly lower (P disinfection of bacterial planktonic cells in source water and ex situ reaction between biofilms and residual chlorine in pipeline networks as hitherto unknown DBP sources in drinking water.

  8. Bacterial cell wall preservation during organic matter diagenesis in sediments off Peru

    DEFF Research Database (Denmark)

    Lomstein, Bente Aagaard; Niggemann, Jutta; Jørgensen, Bo Barker;

    BACTERIAL CELL WALL PRESERVATION DURING ORGANIC MATTER DIAGENESIS IN SEDIMENTS OFF PERU The spatial distribution of total hydrolysable amino acids, total hydrolysable amino sugars and amino acid enantiomers (D- and L-forms) were investigated in surface sediments at 20 stations in the Peru margin: 9...

  9. Bacterial vaginosis (clue cell-positive discharge) : diagnostic, ultra-structural and therapeutic aspects

    NARCIS (Netherlands)

    W.I. van der Meijden (Willem)

    1987-01-01

    textabstractThis thesis deals with several aspects of (abnormal) vaginal discharge, focusing especially on clue cell-positive discharge (bacterial vaginosis, nonspecific vaginitis). It reports data on epidemiology and clinical features, pathogenesis, and treatment of this vaginal disease entity, as

  10. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    R.M. Determann; M. Weisfelt; J. de Gans; A. van der Ende; M.J. Schultz; D. van de Beek

    2006-01-01

    Objective: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. Design: Retrospective study of diagnostic accuracy. Setting and patients: CSF was coll

  11. A portable immunomagnetic cell capture system to accelerate culture diagnosis of bacterial infections.

    Science.gov (United States)

    Singh, Saurabh; Upadhyay, Mohita; Sharma, Jyoti; Gupta, Shalini; Vivekanandan, Perumal; Elangovan, Ravikrishnan

    2016-05-23

    Bacterial infections continue to be a major cause of deaths globally, particularly in resource-poor settings. In the absence of rapid and affordable diagnostic solutions, patients are mostly administered broad spectrum antibiotics leading to antibiotics resistance and poor recovery. Culture diagnosis continues to be a gold standard for diagnosis of bacterial infection, despite its long turnaround time of 24 to 48 h. We have developed a portable immunomagnetic cell capture (iMC(2)) system that allows rapid culture diagnosis of bacterial pathogens. Our approach involves the culture growth of the blood samples in broth media for 6 to 8 h, followed by immunomagnetic enrichment of the target cells using the iMC(2) device. The device comprises a disposable capture chip that has two chambers of 5 ml and 50 μl volume connected through a channel with a manual valve. Bacterial cells bound to antibody coated magnetic nanoparticles are swept from the 5 ml sample chamber into the 50 μl recovery chamber by moving an external magnetic field with respect to the capture chip using a linear positioner. This enables specific isolation and up to 100× enrichment of the target cells. The presence of bacteria in the recovered sample is confirmed visually using a lateral flow immunoassay. The system is demonstrated in buffer and blood samples spiked with S. typhi. The method has high sensitivity (10 CFU ml(-1)), specificity and a rapid turnaround time of less than 7 h, a significant improvement over conventional methods. PMID:27118505

  12. Increased electrical output when a bacterial ABTS oxidizer is used in a microbial fuel cell

    Science.gov (United States)

    Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. The present study examined the formation in culture of an unidentified bacterial oxidant and investigated the ...

  13. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

    Directory of Open Access Journals (Sweden)

    Yihan Lin

    Full Text Available Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS.

  14. Bacterial Cell Surface Adsorption of Rare Earth Elements

    Science.gov (United States)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  15. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  16. Spatial Patterning of Newly-Inserted Material during Bacterial Cell Growth

    Science.gov (United States)

    Ursell, Tristan

    2012-02-01

    In the life cycle of a bacterium, rudimentary microscopy demonstrates that cell growth and elongation are essential characteristics of cellular reproduction. The peptidoglycan cell wall is the main load-bearing structure that determines both cell shape and overall size. However, simple imaging of cellular growth gives no indication of the spatial patterning nor mechanism by which material is being incorporated into the pre-existing cell wall. We employ a combination of high-resolution pulse-chase fluorescence microscopy, 3D computational microscopy, and detailed mechanistic simulations to explore how spatial patterning results in uniform growth and maintenance of cell shape. We show that growth is happening in discrete bursts randomly distributed over the cell surface, with a well-defined mean size and average rate. We further use these techniques to explore the effects of division and cell wall disrupting antibiotics, like cephalexin and A22, respectively, on the patterning of cell wall growth in E. coli. Finally, we explore the spatial correlation between presence of the bacterial actin-like cytoskeletal protein, MreB, and local cell wall growth. Together these techniques form a powerful method for exploring the detailed dynamics and involvement of antibiotics and cell wall-associated proteins in bacterial cell growth.[4pt] In collaboration with Kerwyn Huang, Stanford University.

  17. Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

    Science.gov (United States)

    Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

    2015-12-01

    We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion. PMID:26724085

  18. Inclusion bodies, bacterial cells and compositions containing them and uses thereof

    OpenAIRE

    Veciana Miró, Jaume; Ratera Bastardas, Inmaculada; Díez Gil, César; Villaverde Corrales, Antonio Pedro; Vázquez Gómez, Esther; García Fruitós, Elena

    2008-01-01

    [EN] The present invention relates to an isolated inclusion body comprising a polypeptide, characterised in that such inclusion body is in particulate formo The present invention also refers to a bacterial cell comprising said inclusion body. The present invention additionally refers to a composition comprising said inclusion body and a eukaryotic cell. The present invention moreover refers to a composition comprising said inclusion body and animal or plant tissue. The present invent...

  19. Bacterial glycosidases for the production of universal red blood cells

    DEFF Research Database (Denmark)

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping;

    2007-01-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this techno...

  20. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    Science.gov (United States)

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. PMID:26914280

  1. Electron microscopy study of antioxidant interaction with bacterial cells

    Science.gov (United States)

    Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

    2000-10-01

    To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

  2. Cell wall mechanical properties as measured with bacterial thread made from Bacillus subtilis.

    OpenAIRE

    Mendelson, N H; Thwaites, J J

    1989-01-01

    Engineering approaches used in the study of textile fibers have been applied to the measurement of mechanical properties of bacterial cell walls by using the Bacillus subtilis bacterial thread system. Improved methods have been developed for the production of thread and for measuring its mechanical properties. The best specimens of thread produced from cultures of strain FJ7 grown in TB medium at 20 degrees C varied in diameter by a factor of 1.09 over a 30-mm thread length. The stress-strain...

  3. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  4. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  5. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  6. Induction of delayed-type hypersensitivity by the T cell line specific to bacterial peptidoglycans

    International Nuclear Information System (INIS)

    A T cell line specific for the chemically well-defined peptidoglycan of bacterial cell wall, disaccharide tetrapeptide, was established from Lewis rats immunized with the antigen covalently linked to the autologous rat serum albumin. The antigen specificity was examined with various analogues or derivatives of the peptidoglycan. The cell line was reactive to analogues with the COOH-terminal D-amino acid, but least reactive to those with L-amino acid as COOH terminus. Transferring of the T cell line into X-irradiated normal Lewis rats induced delayed-type hypersensitivity in an antigen specific manner

  7. Mutagenic effect of accelerated heavy ions on bacterial cells

    Science.gov (United States)

    Boreyko, A. V.; Krasavin, E. A.

    2011-11-01

    The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific

  8. Cell fate regulation governed by a repurposed bacterial histidine kinase.

    Directory of Open Access Journals (Sweden)

    W Seth Childers

    2014-10-01

    Full Text Available One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  9. Analysis of gene expression levels in individual bacterial cells without image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  10. Comparison of bacterial cells and amine-functionalized abiotic surfaces as support for Pd nanoparticle synthesis

    DEFF Research Database (Denmark)

    De Corte, Simon; Bechstein, Stefanie; Lokanathan, Arcot R.;

    2013-01-01

    An increasing demand for catalytic Pd nanoparticles has motivated the search for sustainable production methods. An innovative approach uses bacterial cells as support material for synthesizing Pd nanoparticles by reduction of Pd(II) with e.g. hydrogen or formate. Nevertheless, drawbacks of...... on these surfaces was higher than for Pd particles formed on Shewanella oneidensis cells. Smaller Pd nanoparticles generally have better catalytic properties, and previous studies have shown that the particle size can be lowered by increasing the amount of support material used during Pd particle...... materials were visualized by transmission electron microscopy, and their activity was evaluated by catalysis of p-nitrophenol reduction. Surfaces functionalized with 3-aminopropyltriethoxysilane and chitosan were interesting alternatives to bacterial cells, as the catalytic activity of Pd particles formed...

  11. Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods.

    Science.gov (United States)

    Le Blay, Gwenaëlle; Fliss, Ismaïl; Lacroix, Christophe

    2004-11-01

    Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

  12. From Single Cells to Engineered and Explanted Tissues: New Perspectives in Bacterial Infection Biology.

    Science.gov (United States)

    Bergmann, Simone; Steinert, Michael

    2015-01-01

    Cell culture techniques are essential for studying host-pathogen interactions. In addition to the broad range of single cell type-based two-dimensional cell culture models, an enormous amount of coculture systems, combining two or more different cell types, has been developed. These systems enable microscopic visualization and molecular analyses of bacterial adherence and internalization mechanisms and also provide a suitable setup for various biochemical, immunological, and pharmacological applications. The implementation of natural or synthetical scaffolds elevated the model complexity to the level of three-dimensional cell culture. Additionally, several transwell-based cell culture techniques are applied to study bacterial interaction with physiological tissue barriers. For keeping highly differentiated phenotype of eukaryotic cells in ex vivo culture conditions, different kinds of microgravity-simulating rotary-wall vessel systems are employed. Furthermore, the implementation of microfluidic pumps enables constant nutrient and gas exchange during cell cultivation and allows the investigation of long-term infection processes. The highest level of cell culture complexity is reached by engineered and explanted tissues which currently pave the way for a more comprehensive view on microbial pathogenicity mechanisms. PMID:26404465

  13. GROWTH AND METABOLISM OF INDIVIDUAL BACTERIAL CELLS UTILIZING NANOSIMS

    Energy Technology Data Exchange (ETDEWEB)

    NEALSON, H. K.

    2007-08-03

    This work involved the use of the Nano-SIMS Instrument at Lawrence Livermore Laboratory, in an effort to utilize this unique tool for experiments in Biology. The work consisted primarily of experiments to measure in real time, C and N fixation in cyanobacteria. The work revealed a number of the difficulties in using the nano-SIMS approach with biological material, but with collaboration from a number of individuals at USC and LLNL, major progress was made. The collaborators from LLNL were from the Chemistry Group (Dr. Peter Weber), and the Biology Group (Dr. Jennifer Pett-Ridge). In addition, there were a number of other scientists involved from LLNL. The USC group consisted of Dr. K.H. Nealson, the PI on the grant, Dr. R. Popa, a postdoctoral fellow and research associate at USC, Professor Douglas Capone, and Juliet Finze, a graduate student in biology. Two major experiments were done, both of which yielded new and exciting data. (1) We studied nitrogen and carbon fixation in Anabaena, demonstrating that fixation ofN occurred rapidly in the heterocysts, and that the fixed N was transported rapidly and completely to the vegetative cells. C fixation occurred in the vegetative cells, with labeled C remaining in these cells in support of their growth and metabolism. This work was accepted in the ISME Journal (Nature Publication), and published last month. (2) We studied nitrogen and carbon fixation in Trichodesmium, a non-heterocystous cyanobacterium that also fixes nitrogen. Interestingly, the nitrogen fixation was confined to regions within the filaments that seem to be identical to the so-called cyanophycaen granules. The fixed N is then transported to other parts of the cyanobacterium, as judged by movement of the heavy N throughout the filaments. On the basis of these very exciting results, we have applied for funding from the NSF to continue the collaboration with LLNL. The results of both studies were presented in the summer of 2007 at the Gordon Research

  14. Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

    Directory of Open Access Journals (Sweden)

    Marc Daigneault

    Full Text Available Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+ T-cells in PBMC cultures required 'classical' CD14(+ monocytes, which enhanced T-cell activation. CD3(+ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

  15. Lipid II: a central component in bacterial cell wall synthesis and a target for antibiotics.

    Science.gov (United States)

    de Kruijff, Ben; van Dam, Vincent; Breukink, Eefjan

    2008-01-01

    The bacterial cell wall is mainly composed of peptidoglycan, which is a three-dimensional network of long aminosugar strands located on the exterior of the cytoplasmic membrane. These strands consist of alternating MurNAc and GlcNAc units and are interlinked to each other via peptide moieties that are attached to the MurNAc residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane on a polyisoprenoid anchor and one of the key components in the synthesis of peptidoglycan is Lipid II. Being essential for bacterial cell survival, it forms an attractive target for antibacterial compounds such as vancomycin and several lantibiotics. Lipid II consists of one GlcNAc-MurNAc-pentapeptide subunit linked to a polyiosoprenoid anchor 11 subunits long via a pyrophosphate linker. This review focuses on this special molecule and addresses three questions. First, why are special lipid carriers as polyprenols used in the assembly of peptidoglycan? Secondly, how is Lipid II translocated across the bacterial cytoplasmic membrane? And finally, how is Lipid II used as a receptor for lantibiotics to kill bacteria? PMID:19008088

  16. PROCALCITONIN AS A BIOMARKER OF BACTERIAL INFECTION IN SICKLE CELL VASO-OCCLUSIVE CRISIS.

    Directory of Open Access Journals (Sweden)

    Dilip Kumar Patel

    2014-02-01

    Full Text Available Bacterial infection is an important trigger of vaso-occlusive crisis (VOC in sickle cell anaemia (SCA. SCA Patients with VOC have signs of inflammation and it is difficult to diagnose bacterial infection in them. This study was undertaken to evaluate serum procalcitonin (PCT as a biomarker of bacterial infection in acute sickle cell vaso-occlusive crisis. Hundred SCA patients were studied at Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India. SCA was diagnosed by haemoglobin electrophoresis, HPLC and molecular analysis. Patients were divided into 3categories namely Category-A (VOC/ACS with fever but without evidence of bacterial infection-66 patients; Category-B (VOC with fever and documentedbacterial infection-24 patients; and Category-C (Patients in steady statewithout VOC/ACS or fever-10 patients. Investigations like complete blood count, C-reactive protein estimation and PCT measurement was done in all the cases. There was no significant difference in total leucocytes count and C-reactiveprotein values between category A and B. In category A the PCT level was 0.5ng/mL with 87.5% of cases having >2ng/mL. In category C, PCT value was 2ng/mL is indicative of bacterial infection necessitating antimicrobial therapy. Patients with indeterminate PCT value of0.5-2ng/mL, need a repeat PCT estimation or an empirical antibiotic therapyawaiting the availability of microbiological report as deemed necessary.

  17. Convergent development of anodic bacterial communities in microbial fuel cells.

    KAUST Repository

    Yates, Matthew D

    2012-05-10

    Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470±20 mV) and maximum power densities (590±170 mW m(-2)). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.

  18. Mucosal-associated invariant T (MAIT cells: new players in anti-bacterial immunity

    Directory of Open Access Journals (Sweden)

    James E Ussher

    2014-10-01

    Full Text Available Mucosal-associated invariant T (MAIT cells are an innate-like T cell population involved in antibacterial immunity. In humans, MAIT cells are abundant, comprising ~ 10% of the CD8+ T cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariant T cell receptor (Vα7.2-Jα33/12/20 and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MR1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in humans. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in antibacterial immunity, including against E. coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In humans, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with antiretroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including with non-typhoidal Salmonella. In this review we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

  19. DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

    Science.gov (United States)

    Wang, Siyuan

    2012-02-01

    Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

  20. Single-cell-based sensors and synchrotron FTIR spectroscopy: a hybrid system towards bacterial detection.

    Science.gov (United States)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C; Bertozzi, Carolyn; Zhang, Miqin

    2007-09-30

    Microarrays of single macrophage cell-based sensors were developed and demonstrated for potential real-time bacterium detection by synchrotron FTIR microscopy. The cells were patterned on gold electrodes of silicon oxide substrates by a surface engineering technique, in which the gold electrodes were immobilized with fibronectin to mediate cell adhesion and the silicon oxide background was passivated with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. Cell morphology and IR spectra of single, double, and triple cells on gold electrodes exposed to lipopolysaccharide (LPS) of different concentrations were compared to reveal the detection capability of this cell-based sensing platform. The single-cell-based system was found to generate the most significant and consistent IR spectrum shifts upon exposure to LPS, thus providing the highest detection sensitivity. Changes in cell morphology and IR shifts upon cell exposure to LPS were found to be dependent on the LPS concentration and exposure time, which established a method for the identification of LPS concentration and infected cell population. Possibility of using this single-cell system with conventional IR spectroscopy as well as its limitation was investigated by comparing IR spectra of single-cell arrays with gold electrode surface areas of 25, 100, and 400 microm2 using both synchrotron and conventional FTIR spectromicroscopes. This cell-based platform may potentially provide real-time, label-free, and rapid bacterial detection, and allow for high-throughput statistical analyses, and portability. PMID:17560777

  1. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    Science.gov (United States)

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples. PMID:27095488

  2. Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation.

    Science.gov (United States)

    Chaudhari, Snehal N; Mukherjee, Madhumati; Vagasi, Alexandra S; Bi, Gaofeng; Rahman, Mohammad M; Nguyen, Christine Q; Paul, Ligi; Selhub, Jacob; Kipreos, Edward T

    2016-07-11

    Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation. PMID:27404357

  3. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count

    NARCIS (Netherlands)

    Koop, G.; Dik, N.; Nielen, M.; Lipman, L.J.A.

    2010-01-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms,

  4. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kamnev, Alexander A. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation)]. E-mail: aakamnev@ibppm.sgu.ru; Tugarova, Anna V. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Antonyuk, Lyudmila P. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Tarantilis, Petros A. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Kulikov, Leonid A. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Perfiliev, Yurii D. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Polissiou, Moschos G. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Gardiner, Philip H.E. [Division of Chemistry, School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2006-07-28

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with {sup 57}Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

  5. Radiosensitization of hypoxic bacterial cells and animal tumours by membrane active drugs and hyperthermia

    International Nuclear Information System (INIS)

    The present report deals with the results on phenothiazine derivatives such as promethazine (PMZ), trimeprazine (TMZ), trifluoperazine (TFP) and prochlorperazine (PCP) and their comparison with that of chlorpromazine (CPZ). Their efficiency in combination with hyperthermia, radiation and other anti-cancer drugs in treating murine tumors has also been presented herein. In addition, results on bacterial cells dealing with their mechanistic aspects are also included. (author). 57 refs., 27 figures, 13 tables

  6. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    OpenAIRE

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; David H Sherman; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduc...

  7. Bacterial regulatory networks—from self-organizing molecules to cell shape and patterns in bacterial communities

    OpenAIRE

    Hengge, Regine; Sourjik, Victor

    2013-01-01

    The ESF–EMBO Conference on ‘Bacterial Networks' was held in March, 2013. It brought together molecular microbiologists, bacteral systems biologists and synthetic biologists to discuss the architecture, function and dynamics of regulatory networks in bacteria.

  8. Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics.

    Science.gov (United States)

    Rathore, Anurag S; Pathak, Mili; Godara, Avinash

    2016-03-01

    Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G-CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP-HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355-362, 2016. PMID:26588604

  9. Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics.

    Science.gov (United States)

    Rathore, Anurag S; Pathak, Mili; Godara, Avinash

    2016-03-01

    Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G-CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP-HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355-362, 2016.

  10. Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

    Directory of Open Access Journals (Sweden)

    Andressa Vilas Boas Nogueira

    2014-01-01

    Full Text Available The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS of low (CTSL and high (CTSH magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2 and prostaglandin E2 (PGE2 was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG and receptor activator of nuclear factor kappa-B ligand (RANKL were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P<0.05 increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

  11. Mineralization of Iron Oxyhydroxides in the Presence and in the Absence of Bacterial Cells

    Science.gov (United States)

    Châtellier, X.; West, M.; Rose, J.; Fortin, D.; Leppard, G. G.; Ferris, G.

    2001-12-01

    Because of their small size, iron oxides have a large surface area per unit weight ratio and are believed to play an important role as an adsorbing phase in lake sediments for various molecules, including potentially dangerous ones like heavy metals. They have been observed to form in close association with bacterial cells, by oxidation of ferrous ions. It is thus important to determine whether the presence of the bacterial cells can affect the mineralogy and the mesoscopic structure of the Fe-oxides particles, as well as their reactivity towards heavy metals. We synthesized in the lab nanoparticles of Fe-oxides by oxidation of ferrous ions. This was done in the presence and in the absence of various bacterial strains (Escherichia coli, Bacillus subtilis, Pseudomonas Aeruginosa and Bacillus licheniformis) and of inorganic ligands (sulfate, phosphate, silicate). The Fe-oxides particles were then observed by TEM on thin sections and on whole mounts. The chemical composition was estimated by wet chemistry and by EDS. The mineralogy was determined by XRD, SAED and EXAFS. Surface area was investigated by BET. And adsorption of cadmium was also measured at various pH. We observed that the size and the morphology of the particles as well as their mesoscopic spatial organization can be affected by the presence of the cells, whereas the mineralogy is controlled by the chemistry of the solution. The adsorption isotherms of cadmium on the various Fe-oxides will be discussed at the light of these observations.

  12. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  13. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

    Directory of Open Access Journals (Sweden)

    Sandra Schwarz

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  14. Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein

    OpenAIRE

    Blair, Jimmy A.; Xu, Qingping; Childers, W. Seth; Mathews, Irimpan I.; Kern, Justin W.; Eckart, Michael; Deacon, Ashley M.; Shapiro, Lucy

    2013-01-01

    Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus ChpT is an essential mediator within the cell cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (RRs)—CtrA or CpdR—that controls cell cycle progression. To understand how ChpT interacts with multiple...

  15. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti...

  16. Immobilization of cells with nitrilase activity from a thermophilic bacterial strain.

    Science.gov (United States)

    Kabaivanova, L; Dobreva, E; Dimitrov, P; Emanuilova, E

    2005-01-01

    Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.

  17. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines☆

    Science.gov (United States)

    Alberdi, M. Pilar; Dalby, Matthew J.; Rodriguez-Andres, Julio; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

    2012-01-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed ‘tick-only’ viruses inhabiting tick cell lines. PMID:22743047

  18. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines.

    Science.gov (United States)

    Alberdi, M Pilar; Dalby, Matthew J; Rodriguez-Andres, Julio; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2012-06-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

  19. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  20. The periplasmic enzyme, AnsB, of Shigella flexneri modulates bacterial adherence to host epithelial cells.

    Directory of Open Access Journals (Sweden)

    Divya T George

    Full Text Available S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

  1. Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniques

    OpenAIRE

    Wong, Christine Shiang Yee

    2014-01-01

    In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are...

  2. Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

    Science.gov (United States)

    Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-08-30

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme. PMID:23990562

  3. Behind the lines–actions of bacterial type III effector proteins in plant cells

    Science.gov (United States)

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:27526699

  4. Disruption of Bacterial Cells by Photocatalysis of Montmorillonite Supported Titanium Dioxide

    Institute of Scientific and Technical Information of China (English)

    LEI Shaomin; GUO Gaoli; XIONG Bihua; GONG Wenqi; MEI Guangjun

    2009-01-01

    The photo-induced antibacterial capacity of montmorillonite supported titanium dioxide(TiO_2/Mmt for short)was evaluated by using Escherichia coli and Staphylococcus aureus as modal organisms.The bactericidal activity of TiO_2/Mmt was examined by cell viability assay under different illumination modes.Atomic force microscopy(AFM)and total organic carbon/Total nitrogen (TOC/TN)analyses were employed to investigate the mechanism of the photocatalytic bactericidal process qualitatively and quantitatively.The kinetic data show that TiO_2/Mmt has excellent antibac-terial performance,and about 99%of both bacteria cells are inactivated within 75 min illumination.The AFM images demonstrate that the bacterial cells are irreversibly decomposed and some cell components are dissolved.Therefore,the content and phase of carbon and nitrogen in the solution are changed after photocatalytic reaction.

  5. Solid-State NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization

    International Nuclear Information System (INIS)

    Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool. (authors)

  6. Long acting β2-agonist and corticosteroid restore airway glandular cell function altered by bacterial supernatant

    Directory of Open Access Journals (Sweden)

    Nawrocki-Raby Béatrice

    2010-01-01

    Full Text Available Abstract Background Staphylococcus aureus releases virulence factors (VF that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting β2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal combined with a corticosteroid (fluticasone propionate, FP was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. Methods A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. Results When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFα. Conclusions Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting β2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of β2 adrenergic receptor agonist and glucocorticoid.

  7. Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

    Science.gov (United States)

    Helfrich, Stefan; Pfeifer, Eugen; Krämer, Christina; Sachs, Christian Carsten; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina; Frunzke, Julia

    2015-11-01

    Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations.

  8. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    Science.gov (United States)

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  9. Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Hofstadt, M. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Hüttener, M.; Juárez, A. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament de Microbiologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona (Spain); Gomila, G., E-mail: ggomila@ibecbarcelona.eu [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament d' Electronica, Universitat de Barcelona, C/ Marti i Franqués 1, 08028 Barcelona (Spain)

    2015-07-15

    With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates. - Highlights: • Gelatine coatings used to weakly attach bacterial cells onto planar substrates. • Use of the dynamic jumping mode as a non-perturbing bacterial imaging mode. • Nanoscale resolution imaging of unperturbed single living bacterial cells. • Growth and division of single bacteria cells on planar substrates observed.

  10. Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

    OpenAIRE

    Seras Franzoso, Joaquin; Peebo, Karl; Garcia Fruitós, Elena; Vázquez Gómez, Esther; Rinas, Ursula; Villaverde Corrales, Antonio

    2014-01-01

    Altres ajuts: We are indebted CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, Spain) for funding our research on inclusion bodies. Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with...

  11. Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter.

    Science.gov (United States)

    Johansson, Cecilia; Wick, Mary Jo

    2004-02-15

    The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c(+) cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8alpha(-)CD4(-) double negative cells constituted the majority of liver CD11c(+) ( approximately 85%) with few cells expressing CD8alpha or CD4. Flow cytometry analysis of freshly isolated CD11c(+) cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c(+) MHC class II(high) cells had a greater capacity to internalize Salmonella relative to CD11c(+) MHC class II(low) cells. Moreover, both CD8alpha(-) and CD8alpha(+) liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c(+) cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c(+) cells infected with Salmonella expressing GFP showed that both CD8alpha(-) and CD8alpha(+) DC produced IL-12p40 and TNF-alpha. The majority of cytokine-positive cells did not contain bacteria (GFP(-)) whereas only a minor fraction of cytokine-positive cells were GFP(+). Furthermore, only approximately 30-50% of liver DC containing bacteria (GFP(+)) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4(+) and CD8(+) T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.

  12. Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments.

    Science.gov (United States)

    Yang, Desirée C; Blair, Kris M; Salama, Nina R

    2016-03-01

    Bacteria display an abundance of cellular forms and can change shape during their life cycle. Many plausible models regarding the functional significance of cell morphology have emerged. A greater understanding of the genetic programs underpinning morphological variation in diverse bacterial groups, combined with assays of bacteria under conditions that mimic their varied natural environments, from flowing freshwater streams to diverse human body sites, provides new opportunities to probe the functional significance of cell shape. Here we explore shape diversity among bacteria, at the levels of cell geometry, size, and surface appendages (both placement and number), as it relates to survival in diverse environments. Cell shape in most bacteria is determined by the cell wall. A major challenge in this field has been deconvoluting the effects of differences in the chemical properties of the cell wall and the resulting cell shape perturbations on observed fitness changes. Still, such studies have begun to reveal the selective pressures that drive the diverse forms (or cell wall compositions) observed in mammalian pathogens and bacteria more generally, including efficient adherence to biotic and abiotic surfaces, survival under low-nutrient or stressful conditions, evasion of mammalian complement deposition, efficient dispersal through mucous barriers and tissues, and efficient nutrient acquisition. PMID:26864431

  13. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  14. The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

    Directory of Open Access Journals (Sweden)

    Oliveira S.C.

    1998-01-01

    Full Text Available Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

  15. Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments.

    Science.gov (United States)

    Yang, Desirée C; Blair, Kris M; Salama, Nina R

    2016-03-01

    Bacteria display an abundance of cellular forms and can change shape during their life cycle. Many plausible models regarding the functional significance of cell morphology have emerged. A greater understanding of the genetic programs underpinning morphological variation in diverse bacterial groups, combined with assays of bacteria under conditions that mimic their varied natural environments, from flowing freshwater streams to diverse human body sites, provides new opportunities to probe the functional significance of cell shape. Here we explore shape diversity among bacteria, at the levels of cell geometry, size, and surface appendages (both placement and number), as it relates to survival in diverse environments. Cell shape in most bacteria is determined by the cell wall. A major challenge in this field has been deconvoluting the effects of differences in the chemical properties of the cell wall and the resulting cell shape perturbations on observed fitness changes. Still, such studies have begun to reveal the selective pressures that drive the diverse forms (or cell wall compositions) observed in mammalian pathogens and bacteria more generally, including efficient adherence to biotic and abiotic surfaces, survival under low-nutrient or stressful conditions, evasion of mammalian complement deposition, efficient dispersal through mucous barriers and tissues, and efficient nutrient acquisition.

  16. Emulsification efficiency of adsorbed chitosan for bacterial cells accumulation at the oil-water interface.

    Science.gov (United States)

    Archakunakorn, Somwit; Charoenrat, Nattapat; Khamsakhon, Somruethai; Pongtharangkul, Thunyarat; Wongkongkatep, Pravit; Suphantharika, Manop; Wongkongkatep, Jirarut

    2015-04-01

    The use of bacterial cell or biocatalyst for industrial synthetic chemistry is on the way of significant growth since the biocatalyst requires low energy input compared to the chemical synthesis and can be considered as a green technology. However, majority of natural bacterial cell surface is hydrophilic which allows poor access to the hydrophobic substrate or product. In this study, Escherichia coli (E. coli) as a representative of hydrophilic bacterial cells were accumulated at the oil-water interface after association with chitosan at a concentration range of 0.75-750 mg/L. After association with negatively charged E coli having a ζ potential of -19.9 mV, a neutralization of positively charged chitosan occurred as evidenced by an increase in the ζ potential value of the mixtures with increasing chitosan concentration up to +3.5 mV at 750 mg/L chitosan. Both emulsification index and droplet size analysis revealed that chitosan-E. coli system is an excellent emulsion stabilizer to date because the threshold concentration was as low as 7.5 mg/L or 0.00075% w/v. A dramatic increase in the surface hydrophobicity of the E. coli as evidenced by an increase in contact angle from 19 to 88° with increasing chitosan concentration from 0 to 750 mg/L, respectively, resulted in an increase in the stability of oil-in-water emulsions stabilized by chitosan-E. coli system. The emulsion was highly stable even the emulsification was performed under 20% salt condition, or temperature ranged between 20 and 50 °C. Emulsification was failed when the oil volume fraction was higher than 0.5, indicating that no phase inversion occurred. The basic investigation presented in this study is a crucial platform for its application in biocatalyst industry and bioremediation of oil spill. PMID:25341365

  17. Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Husen Zhang

    Full Text Available Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

  18. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric;

    2010-01-01

    Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial....... From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas...... fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly...

  19. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre;

    2005-01-01

    Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells....... By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r......RNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria...

  20. Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

    Science.gov (United States)

    Bravo-Angel, A M; Gloeckler, V; Hohn, B; Tinland, B

    1999-09-01

    Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins. PMID:10482518

  1. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    Science.gov (United States)

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  2. Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition

    Science.gov (United States)

    Tang, Hua; Li, Wen-Chao; Wu, Hao; Ding, Hui

    2016-01-01

    Owing to the abuse of antibiotics, drug resistance of pathogenic bacteria becomes more and more serious. Therefore, it is interesting to develop a more reasonable way to solve this issue. Because they can destroy the bacterial cell structure and then kill the infectious bacterium, the bacterial cell wall lyases are suitable candidates of antibacteria sources. Thus, it is urgent to develop an accurate and efficient computational method to predict the lyases. Based on the consideration, in this paper, a set of objective and rigorous data was collected by searching through the Universal Protein Resource (the UniProt database), whereafter a feature selection technique based on the analysis of variance (ANOVA) was used to acquire optimal feature subset. Finally, the support vector machine (SVM) was used to perform prediction. The jackknife cross-validated results showed that the optimal average accuracy of 84.82% was achieved with the sensitivity of 76.47% and the specificity of 93.16%. For the convenience of other scholars, we built a free online server called Lypred. We believe that Lypred will become a practical tool for the research of cell wall lyases and development of antimicrobial agents. PMID:27437396

  3. Applications of whole-cell bacterial sensors in biotechnology and environmental science

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Kiyohito [Osaka Univ., Suita (Japan). Graduate School of Pharmaceutical Sciences

    2007-01-15

    Biosensors have major advantages over chemical or physical analyses with regard to specificity, sensitivity, and portability. Recently, many types of whole-cell bacterial biosensors have been developed using recombinant DNA technology. The bacteria are genetically engineered to respond to the presence of chemicals or physiological stresses by synthesizing a reporter protein, such as luciferase, {beta}-galactosidase, or green fluorescent protein. In addition to an overview of conventional biosensors, this minireview discusses a novel type of biosensor using a photosynthetic bacterium as the sensor strain and the crtA gene, which is responsible for carotenoid synthesis, as the reporter. Since bacteria possess a wide variety of stress-response mechanisms, including antioxidation, heat-shock responses, nutrient-starvation, and membrane-damage responses, DNA response elements for several stress-response proteins can be fused with various reporter genes to construct a versatile set of bacterial biosensors for a variety of analytes. Portable biosensors for on-site monitoring have been developed using a freeze-dried biosensing strain, and cell array biosensors have been designed for high-throughput analysis. Moreover, in the future, the use of single-cell biosensors will permit detailed analyses of samples. Signals from such sensors could be detected with digital imaging, epifluorescence microscopy, and/or flow cytometry. (orig.)

  4. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

    Science.gov (United States)

    Muir, Elizabeth M; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W; Keynes, Roger J; Rogers, John H

    2010-01-15

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

  5. Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition.

    Science.gov (United States)

    Chen, Xin-Xin; Tang, Hua; Li, Wen-Chao; Wu, Hao; Chen, Wei; Ding, Hui; Lin, Hao

    2016-01-01

    Owing to the abuse of antibiotics, drug resistance of pathogenic bacteria becomes more and more serious. Therefore, it is interesting to develop a more reasonable way to solve this issue. Because they can destroy the bacterial cell structure and then kill the infectious bacterium, the bacterial cell wall lyases are suitable candidates of antibacteria sources. Thus, it is urgent to develop an accurate and efficient computational method to predict the lyases. Based on the consideration, in this paper, a set of objective and rigorous data was collected by searching through the Universal Protein Resource (the UniProt database), whereafter a feature selection technique based on the analysis of variance (ANOVA) was used to acquire optimal feature subset. Finally, the support vector machine (SVM) was used to perform prediction. The jackknife cross-validated results showed that the optimal average accuracy of 84.82% was achieved with the sensitivity of 76.47% and the specificity of 93.16%. For the convenience of other scholars, we built a free online server called Lypred. We believe that Lypred will become a practical tool for the research of cell wall lyases and development of antimicrobial agents. PMID:27437396

  6. Role of Sulfhydryl Sites on Bacterial Cell Walls in the Biosorption, Mobility and Bioavailability of Mercury and Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Myneni, Satish C.; Mishra, Bhoopesh; Fein, Jeremy

    2009-04-01

    The goal of this exploratory study is to provide a quantitative and mechanistic understanding of the impact of bacterial sulfhydryl groups on the bacterial uptake, speciation, methylation and bioavailability of Hg and redox changes of uranium. The relative concentration and reactivity of different functional groups present on bacterial surfaces will be determined, enabling quantitative predictions of the role of biosorption of Hg under the physicochemical conditions found at contaminated DOE sites.The hypotheses we propose to test in this investigation are as follows- 1) Sulfhydryl groups on bacterial cell surfaces modify Hg speciation and solubility, and play an important role, specifically in the sub-micromolar concentration ranges of metals in the natural and contaminated systems. 2) Sulfhydryl binding of Hg on bacterial surfaces significantly influences Hg transport into the cell and the methylation rates by the bacteria. 3) Sulfhydryls on cell membranes can interact with hexavalent uranium and convert to insoluble tetravalent species. 4) Bacterial sulfhydryl surface groups are inducible by the presence of metals during cell growth. Our studies focused on the first hypothesis, and we examined the nature of sulfhydryl sites on three representative bacterial species: Bacillus subtilis, a common gram-positive aerobic soil species; Shewanella oneidensis, a facultative gram-negative surface water species; and Geobacter sulfurreducens, an anaerobic iron-reducing gram-negative species that is capable of Hg methylation; and at a range of Hg concentration (and Hg:bacterial concentration ratio) in which these sites become important. A summary of our findings is as follows- Hg adsorbs more extensively to bacteria than other metals. Hg adsorption also varies strongly with pH and chloride concentration, with maximum adsorption occurring under circumneutral pH conditions for both Cl-bearing and Cl-free systems. Under these conditions, all bacterial species tested exhibit

  7. Microarray Analysis of Human Vascular Smooth Muscle Cell Responses to Bacterial Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Joe Minta

    2007-01-01

    Full Text Available Accumulating evidence suggest a causal role of bacterial and viral infections in atherogenesis. Bacterial lipopolysaccharide (LPS has been shown to stimulate resting vascular smooth muscle cells (SMC with the production of inflammatory cytokines and modulation of quiescent cells to the proliferative and synthetic phenotype. To comprehensively identify biologically important genes associated with LPS-induced SMC phenotype modulation, we compared the transcriptomes of quiescent human coronary artery SMC and cells treated with LPS for 4 and 22 h. The SMCs responded robustly to LPS treatment by the differential regulation of several genes involved in chromatin remodeling, transcription regulation, translation, signal transduction, metabolism, host defense, cell proliferation, apoptosis, matrix formation, adhesion and motility and suggest that the induction of clusters of genes involved in cell proliferation, migration and ECM production may be the main force that drives the LPS-induced phenotypic modulation of SMC rather than the differential expression of a single gene or a few genes. An interesting observation was the early and dramatic induction of four tightly clustered interferon-induced genes with tetratricopeptide repeats (IFIT1, 2, 4, 5. siRNA knock-down of IFIT1 in SMC was found to be associated with a remarkable up-regulation of TP53, CDKN1A and FOS, suggesting that IFIT1 may play a role in cell proliferation. Our data provide a comprehensive list of genes involved in LPS biology and underscore the important role of LPS in SMC activation and phenotype modulation which is a pivotal event in the onset of atherogenesis.

  8. Accuracy of the automated cell counters for management of spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Oliviero Riggio; Stefania Angeloni; Antonella Parente; Cinzia Leboffe; Giorgio Pinto; Teresa Aronne; Manuela Merli

    2008-01-01

    AIM: To evaluate the accuracy of automated blood cell counters for ascitic polymorphonuclear (PMN)determination for: (1) diagnosis,(2) efficacy of the ongoing antibiotic therapy,and (3) resolution of spontaneous bacterial peritonitis (SBP).METHODS: One hundred and twelve ascitic fluid samples were collected from 52 consecutive cirrhotic patients,16 of them with SBR The agreement between the manual and the automated method for PMN count was assessed.The sensitivity/specificity and the positive/negative predictive value of the automated blood cell counter were also calculated by considering the manual method as the "gold standard"RESULTS: The mean + SD of the difference between manual and automated measurements was 7.8±58cells/mm3,while the limits of agreement were +124 cells/mm3 [95% confidence interval (CI): +145 to +103] and -108 cells/mm3 (95% CI: -87 to -129).The automated cell counter had a sensitivity of 100% and a specificity of 97.7% in diagnosing SBP,and a sensitivity of 91% and a specificity of 100% for the efficacy of the ongoing antibiotic therapy.The two methods showed a complete agreement for the resolution of infection.CONCLUSION: Automated cell counters not only have a good diagnostic accuracy,but are also very effective in monitoring the antibiotic treatment in patients with SBP.Because of their quicker performance,they should replace the manual counting for PMN determination in the ascitic fluid of patients with SBR

  9. Peripheral T Cell Apoptosis and Its Role in Generalized Bacterial Infections: A Minireview.

    Science.gov (United States)

    Chernykh, Helen R.; Norkin, Maxim N.; Leplina, Olga Yu.; Khonina, Nataliya A.; Tihonova, Marina A.; Ostanin, Alexander A.

    2001-07-01

    In the present review we have attempted to analyze recent findings concerning apoptosis of mature peripheral T cells. The great attention is made to the factors underlying resistance or sensitivity of mature T lymphocytes to activation-induced cell death. The role of preactivation and altered costimulation is discussed in this regard. Besides, the possible role of cytokines in the modulation of T cell apoptosis is emphasized. Particular attention is paid to the studies of apoptosis disorders in the pathogenesis of generalized bacterial infections. In this connection some own results are summarized as well. To characterize T cell death and its role in the pathogenesis of bacterial infections an anti-CD3-mAb or Con A-induced apoptosis in patients with severe and generalized forms of surgical infections have been investigated. We have found a significant increase of activation-induced lymphocyte apoptosis and a high level of apoptosis in freshly isolated lymphocytes in patients with surgical infections. Alternatively, peripheral blood mononuclear cells from surgical patients without infectious complications did not exhibit a marked enhancement of activation-induced cell death. Activation-induced T cell death in surgical infections appeared to be Fas-dependent, involved reactive oxygen intermediates and was partly prevented by pro-inflammatory cytokines, among which IL-2 exhibited the most pronounced anti-apoptotic activity. Likewise, APACHE II score, as a marker of the infection severity, directly correlated with a rate of activation-induced T cell apoptosis. Accelerated T cell apoptosis at the early stage of infection was revealed in survivors and non-survivors, that appears to designate a common pathway for the restriction of systemic inflammation. At the late stage of infection altered T cell apoptosis could account for different outcomes, since the patients with lethal outcome showed 2-fold increase in activation-induced cell death compared to the opposite group

  10. Regulatory T cell suppressive potency dictates the balance between bacterial proliferation and clearance during persistent Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Tanner M Johanns

    Full Text Available The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3-4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3(GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3(+ Treg suppressive potency. In complementary experiments using Foxp3(DTR mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

  11. Simultaneous selection of soil electroactive bacterial communities associated to anode and cathode in a two-chamber Microbial Fuel Cell

    Science.gov (United States)

    Chiellini, Carolina; Bacci, Giovanni; Fani, Renato; Mocali, Stefano

    2016-04-01

    Different bacteria have evolved strategies to transfer electrons over their cell surface to (or from) their extracellular environment. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES) such as Microbial Fuel Cells (MFCs). In MFC research the biological reactions at the cathode have long been a secondary point of interest. However, bacterial biocathodes in MFCs represent a potential advantage compared to traditional cathodes, for both their low costs and their low impact on the environment. The main challenge in biocathode set-up is represented by the selection of a bacterial community able to efficiently accept electrons from the electrode, starting from an environmental matrix. In this work, a constant voltage was supplied on a two-chamber MFC filled up with soil over three weeks in order to simultaneously select an electron donor bacterial biomass on the anode and an electron acceptor biomass on the cathode, starting from the same soil. Next Generation Sequencing (NGS) analysis was performed to characterize the bacterial community of the initial soil, in the anode, in the cathode and in the control chamber not supplied with any voltage. Results highlighted that both the MFC conditions and the voltage supply affected the soil bacterial communities, providing a selection of different bacterial groups preferentially associated to the anode (Betaproteobacteria, Bacilli and Clostridia) and to the cathode (Actinobacteria and Alphaproteobacteria). These results confirmed that several electroactive bacteria are naturally present within a top soil and, moreover, different soil bacterial genera could provide different electrical properties.

  12. Response Mechanisms of Bacterial Degraders to Environmental Contaminants on the Level of Cell Walls and Cytoplasmic Membrane

    Directory of Open Access Journals (Sweden)

    Slavomíra Murínová

    2014-01-01

    Full Text Available Bacterial strains living in the environment must cope with the toxic compounds originating from humans production. Surface bacterial structures, cell wall and cytoplasmic membrane, surround each bacterial cell and create selective barriers between the cell interior and the outside world. They are a first site of contact between the cell and toxic compounds. Organic pollutants are able to penetrate into cytoplasmic membrane and affect membrane physiological functions. Bacteria had to evolve adaptation mechanisms to counteract the damage originated from toxic contaminants and to prevent their accumulation in cell. This review deals with various adaptation mechanisms of bacterial cell concerning primarily the changes in cytoplasmic membrane and cell wall. Cell adaptation maintains the membrane fluidity status and ratio between bilayer/nonbilayer phospholipids as well as the efflux of toxic compounds, protein repair mechanisms, and degradation of contaminants. Low energy consumption of cell adaptation is required to provide other physiological functions. Bacteria able to survive in toxic environment could help us to clean contaminated areas when they are used in bioremediation technologies.

  13. Probing interaction of Gram-positive and Gram-negative bacterial cells with ZnO nanorods

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Aanchal; Bhargava, Richa; Poddar, Pankaj, E-mail: p.poddar@ncl.res.in

    2013-04-01

    In the present work, the physiological effects of the ZnO nanorods on the Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Aerobacter aerogenes) bacterial cells have been studied. The analysis of bacterial growth curves for various concentrations of ZnO nanorods indicates that Gram positive and Gram negative bacterial cells show inhibition at concentrations of ∼ 64 and ∼ 256 μg/mL respectively. The marked difference in susceptibility towards nanorods was also validated by spread plate and disk diffusion methods. In addition, the scanning electron micrographs show a clear damage to the cells via changed morphology of the cells from rod to coccoid etc. The confocal optical microscopy images of these cells also demonstrate the reduction in live cell count in the presence of ZnO nanorods. These, results clearly indicate that the antibacterial activity of ZnO nanorods is higher towards Gram positive bacterium than Gram negative bacterium which indicates that the structure of the cell wall might play a major role in the interaction with nanostructured materials and shows high sensitivity to the particle concentration. Highlights: ► Effect of ZnO nanorods on the growth cycles of four bacterial strains. ► A relation has been established between growth rate of bacteria and concentration. ► Serious damage in the morphology of bacterial cells in the presence of ZnO nanorods. ► Microscopic studies to see the time dependent effect on bacterial cells.

  14. Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus

    OpenAIRE

    Hayashi, Ikuko; Oyama, Takuji; Morikawa, Kosuke

    2001-01-01

    Proper placement of the bacterial cell division site requires the site-specific inactivation of other potential division sites. In Escherichia coli, selection of the correct mid-cell site is mediated by the MinC, MinD and MinE proteins. To clarify the functional role of the bacterial cell division inhibitor MinD, which is a membrane-associated ATPase that works as an activator of MinC, we determined the crystal structure of a Pyrococcus furiosus MinD homologue complexed with a substrate analo...

  15. Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

    OpenAIRE

    Naz, Neveda; Mills, Dominic C.; Wren, Brendan W.; Dorrell, Nick

    2013-01-01

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusi...

  16. Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

    Science.gov (United States)

    Kulkarni, A D; Fanslow, W C; Drath, D B; Rudolph, F B; Van Buren, C T

    1986-02-01

    Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge. PMID:3947217

  17. Spatiotemporal development of the bacterial community in a tubular longitudinal microbial fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Rae; Premier, Giuliano C. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Advnaced Technology; Beecroft, Nelli J.; Avignone-Rossa, Claudio [Surrey Univ., Guildford (United Kingdom). Microbial Sciences; Varcoe, John R.; Slade, Robert C.T. [Surrey Univ., Guildford (United Kingdom). Chemical Sciences; Dinsdale, Richard M.; Guwy, Alan J. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Health, Sport and Science; Thumser, Alfred [Surrey Univ., Guildford (United Kingdom). Biochemical Sciences

    2011-05-15

    The spatiotemporal development of a bacterial community in an exoelectrogenic biofilm was investigated in sucrose-fed longitudinal tubular microbial fuel cell reactors, consisting of two serially connected modules. The proportional changes in the microbial community composition were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing in order to relate them to the performance and stability of the bioelectrochemical system. The reproducibility of duplicated reactors, evaluated by cluster analysis and Jaccard's coefficient, shows 80-90% similarity in species composition. Biofilm development through fed-batch start-up and subsequent stable continuous operation results in a population shift from {gamma}-Proteobacteria- and Bacteroidetes- to Firmicutes-dominated communities, with other diverse species present at much lower relative proportions. DGGE patterns were analysed by range-weighted richness (Rr) and Pareto-Lorenz evenness distribution curves to investigate the evolution of the bacterial community. The first modules shifted from dominance by species closely related to Bacteroides graminisolvens, Raoultella ornithinolytica and Klebsiella sp. BM21 at the start of continuous-mode operation to a community dominated by Paludibacter propionicigenes-, Lactococcus sp.-, Pantoea agglomerans- and Klebsiella oxytoca-related species with stable power generation (6.0 W/m{sup 3}) at day 97. Operational strategies that consider the dynamics of the population will provide useful parameters for evaluating system performance in the practical application of microbial fuel cells. (orig.)

  18. Nutritional stress induces exchange of cell material and energetic coupling between bacterial species.

    Science.gov (United States)

    Benomar, Saida; Ranava, David; Cárdenas, María Luz; Trably, Eric; Rafrafi, Yan; Ducret, Adrien; Hamelin, Jérôme; Lojou, Elisabeth; Steyer, Jean-Philippe; Giudici-Orticoni, Marie-Thérèse

    2015-02-23

    Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell-cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner (D. vulgaris) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction.

  19. Analysis of a stochastic model for bacterial growth and the lognormality in the cell-size distribution

    CERN Document Server

    Yamamoto, Ken

    2016-01-01

    This paper theoretically analyzes a phenomenological stochastic model for bacterial growth. This model comprises cell divisions and linear growth of cells, where growth rates and cell cycles are drawn from lognormal distributions. We derive that the cell size is expressed as a sum of independent lognormal variables. We show numerically that the quality of the lognormal approximation greatly depends on the distributions of the growth rate and cell cycle. Furthermore, we show that actual parameters of the growth rate and cell cycle take values which give good lognormal approximation, so the experimental cell-size distribution is in good agreement with a lognormal distribution.

  20. Analysis of a Stochastic Model for Bacterial Growth and the Lognormality of the Cell-Size Distribution

    Science.gov (United States)

    Yamamoto, Ken; Wakita, Jun-ichi

    2016-07-01

    This paper theoretically analyzes a phenomenological stochastic model for bacterial growth. This model comprises cell division and the linear growth of cells, where growth rates and cell cycles are drawn from lognormal distributions. We find that the cell size is expressed as a sum of independent lognormal variables. We show numerically that the quality of the lognormal approximation greatly depends on the distributions of the growth rate and cell cycle. Furthermore, we show that actual parameters of the growth rate and cell cycle take values that give a good lognormal approximation; thus, the experimental cell-size distribution is in good agreement with a lognormal distribution.

  1. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    Science.gov (United States)

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  2. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    Science.gov (United States)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  3. Functional heterogeneity in CD4+ T cell responses against a bacterial pathogen

    Directory of Open Access Journals (Sweden)

    Ashley eViehmann Milam

    2015-12-01

    Full Text Available To investigate how CD4+ T cells function against a bacterial pathogen, we generated a Listeria monocytogenes-specific CD4+ T cell model. In this system, two TCRtg mouse lines, LLO56 and LLO118 recognize the same immunodominant epitope (LLO190-205 of Listeria monocytogenes and have identical in vitro responses. However, in vivo LLO56 and LLO118 display vastly different responses during both primary and secondary infection. LLO118 dominates in the primary response and in providing CD8 T cell help. LLO56 predominates in the secondary response. We have also shown that both specific (TCR-mediated and nonspecific stimuli (bypassing the TCR elicit distinct responses from the two transgenics, leading us to conclude that the strength of self-pMHC signaling during development tightly dictates the cell’s future response in the periphery. Herein, we review our findings in this transfer system, focusing on the contribution of the immunomodulatory molecule CD5 and the importance of self-interaction in peripheral maintenance of the cell. We also discuss the manner in which individual TCR affinities to foreign and self-pMHC contribute to the outcome of an immune response; our assertion is that there exists a spectrum of possible T cell responses to recognition of cognate antigen during infection, adding immense diversity to the immune system’s response to pathogens.

  4. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    Science.gov (United States)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  5. Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jennifer C Regan

    2013-10-01

    Full Text Available Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of

  6. Colour removal from aqueous solutions of metal-complex azo dyes using bacterial cells of Shewanella strain J18 143.

    Science.gov (United States)

    Li, Tie; Guthrie, James Thomas

    2010-06-01

    The decoloration treatment of textile dye effluents through biodegradation, using bacterial cells, has been studied as a possible means of solving some of the problems that are associated with the pollution of water sources by colorants. In this paper, the use of whole bacterial cells of Shewanella J18 143 for the reduction of aqueous solutions of selected mono-azo, metal-complex dyes, namely Irgalan Grey GLN, Irgalan Black RBLN and Irgalan Blue 3GL, was investigated. The effects of temperature, pH and dye concentration on colour removal were also investigated and shown to be important. The operative conditions for the removal of colour were 30 degrees C, at pH 6.8, with a final dye concentration of 0.12 g/L in the colour reduction system. This study provides an extension to the application of Shewanella strain J18 143 bacterial cells in the decoloration of textile wastewaters. PMID:20167478

  7. Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

    Directory of Open Access Journals (Sweden)

    Watson CY

    2015-06-01

    Full Text Available Christa Y Watson, Ramon M Molina, Andressa Louzada, Kimberly M Murdaugh, Thomas C Donaghey, Joseph D BrainCenter for Nanotechnology and Nanotoxicology, Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USABackground: Zinc oxide engineered nanoparticles (ZnO ENPs have potential as nanomedicines due to their inherent properties. Studies have described their pulmonary impact, but less is known about the consequences of ZnO ENP interactions with the liver. This study was designed to describe the effects of ZnO ENPs on the liver and Kupffer cells after intravenous (IV administration.Materials and methods: First, pharmacokinetic studies were conducted to determine the tissue distribution of neutron-activated 65ZnO ENPs post-IV injection in Wistar Han rats. Then, a noninvasive in vivo method to assess Kupffer cell phagosomal motility was employed using ferromagnetic iron particles and magnetometry. We also examined whether prior IV injection of ZnO ENPs altered Kupffer cell bactericidal activity on circulating Pseudomonas aeruginosa. Serum and liver tissues were collected to assess liver-injury biomarkers and histological changes, respectively.Results: We found that the liver was the major site of initial uptake of 65ZnO ENPs. There was a time-dependent decrease in tissue levels of 65Zn in all organs examined, reflecting particle dissolution. In vivo magnetometry showed a time-dependent and transient reduction in Kupffer cell phagosomal motility. Animals challenged with P. aeruginosa 24 hours post-ZnO ENP injection showed an initial (30 minutes delay in vascular bacterial clearance. However, by 4 hours, IV-injected bacteria were cleared from the blood, liver, spleen, lungs, and kidneys. Seven days post-ZnO ENP injection, creatine phosphokinase and aspartate aminotransferase levels in serum were significantly increased. Histological evidence of

  8. Micro Corona Ionizer as an Ozone Source for Bacterial Cell Lysis

    Science.gov (United States)

    Lee, Eun-Hee; Lim, Hyun Jeong; Chua, Beelee; Son, Ahjeong

    2015-04-01

    DNA extraction is a critical process of DNA assays including polymerase chain reaction (PCR), microarrays, molecular cloning, and DNA hybridization which has been well established and can be implemented by commercial kits. DNA extraction involves cell lysis, precipitation, and purification through the combination of physical and chemical processes. Cell lysis is essential to high DNA recovery yield which can be achieved via a variety of physical, chemical, and enzymatic methods. However, these methods were originally developed for bioassays that were labor intensive, time consuming, and vulnerable to contamination and inhibition. Here, we proposed to employ a micro corona ionizer as an ozone source to lyse bacterial cells. Ozone has been well known and used as a disinfectant which allows cell lysis and DNA extraction. Previously, we have shown that a micro corona ionizer is capable of generating a significant amount of ozone. In this study, we employed the micro corona ionizer for the bacterial cell lysis which consists of a 50 μm diameter cantilever wire as the discharge cathode and a 50 μm thick copper foil as anode. Applied voltages varied from 1900 to 2200 V with corresponding corona currents from 16 to 28 μA. The resultant ozone (concentration > 0.14 ppm) generated from the micro corona ionizer was bubbled into the sample via a miniature pump. We demonstrated the cell lysis of Pseudomonas putida as the target bacterium using the micro corona ionizer. At a flow rate of 38 ml/min and applied corona voltage of 2000 V, 98.5 ± 0.2% lysis (normalized to sonication result) was achieved after 10 min. In comparison, untreated and air-treated samples showed normalized % lysis of 11.9 ± 2.4 and 36.1 ± 1.7%, respectively. We also showed that the cell lysis efficiency could be significantly increased by increasing the flow rate and the applied corona voltage. By comparing the experimental results for continuous and pulsed treatment, we verified that the percentage of

  9. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens

    Science.gov (United States)

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M. Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

    2014-01-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72 h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen–pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence

  10. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens.

    Science.gov (United States)

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2014-06-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen-pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.

  11. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  12. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

    Science.gov (United States)

    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A

    2007-01-01

    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  13. Cell resistant zwitterionic polyelectrolyte coating promotes bacterial attachment: an adhesion contradiction.

    Science.gov (United States)

    Martinez, Jessica S; Kelly, Kristopher D; Ghoussoub, Yara E; Delgado, Jose D; Keller Iii, Thomas C S; Schlenoff, Joseph B

    2016-04-22

    Polymers of various architectures with zwitterionic functionality have recently been shown to effectively suppress nonspecific fouling of surfaces by proteins and prokaryotic (bacteria) or eukaryotic (mammalian) cells as well as other microorganisms and environmental contaminants. In this work, zwitterionic copolymers were used to make thin coatings on substrates with the layer-by-layer method. Polyelectrolyte multilayers, PEMUs, were built with [poly(allylamine hydrochloride)], PAH, and copolymers of acrylic acid and either the AEDAPS zwitterionic group 3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate (PAA-co-AEDAPS), or benzophenone (PAABp). Benzophenone allowed the PEMU to be toughened by photocrosslinking post-deposition. The attachment of two mammalian cell lines, rat aortic smooth muscle (A7r5) and mouse fibroblasts (3T3), and the biofilm-forming Gram-negative bacteria Escherichia coli was studied on PEMUs terminated with PAA-co-AEDAPS. Consistent with earlier studies, it is shown that PAH/PAA-co-AEDAPS PEMUs resist the adhesion of mammalian cells, but, contrary to our initial hypothesis, are bacterial adhesive and significantly so after maximizing the surface presentation of PAA-co-AEDAPS. This unexpected contrast in the adhesive behavior of prokaryotic and eukaryotic cells is explained by differences in adhesion mechanisms as well as different responses to the topology and morphology of the multilayer surface. PMID:26872345

  14. Bacterial capsular polysaccharide prevents the onset of asthma through T-cell activation.

    Science.gov (United States)

    Johnson, Jenny L; Jones, Mark B; Cobb, Brian A

    2015-04-01

    Over the last four decades, increases in the incidence of immune-mediated diseases in the Western world have been linked to changes in microbial exposure. It is becoming increasingly clear that the normal microbiota in the gut can profoundly alter susceptibility to a wide range of diseases, such as asthma, in which immune homeostasis is disrupted, yet the mechanisms governing this microbial influence remains poorly defined. In this study, we show that gastrointestinal exposure to PSA, a capsular polysaccharide derived from the commensal bacterium Bacteroides fragilis, significantly limits susceptibility to the induction of experimental asthma. We report that direct treatment of mice with PSA generates protection from asthma, and this effect can be given to a naïve recipient by adoptive transfer of CD4(+) T cells from PSA-exposed mice. Remarkably, we found that these PSA-induced T cells are not canonical FoxP3(+) regulatory T cells, but that they potently inhibit both Th1 and Th2 models of asthma in an IL-10-dependent fashion. These findings reveal that bacterial polysaccharides link the microbiota with the peripheral immune system by activating CD4(+)Foxp3(-) T cells upon exposure in the gut, and they facilitate resistance to unnecessary inflammatory responses via the production of IL-10. PMID:25347992

  15. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    Directory of Open Access Journals (Sweden)

    Vera Kozjak-Pavlovic

    2009-10-01

    Full Text Available The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m. Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m loss and apoptosis, demonstrating that dissipation of DeltaPsi(m is a requirement for cell death caused by neisserial infection.

  16. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    International Nuclear Information System (INIS)

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs

  17. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  18. Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell.

    Science.gov (United States)

    Lindsay, M R; Webb, R I; Strous, M; Jetten, M S; Butler, M K; Forde, R J; Fuerst, J A

    2001-06-01

    The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces maris and "Candidatus Brocadia anammoxidans" was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellula marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces maris is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising ("anammox" process) chemoautotroph "Candidatus Brocadia anammoxidans" the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anammoxosome. In Gemmata obscuriglobus, the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria. PMID:11491082

  19. Direct measurement of cell wall stress-stiffening and turgor pressure in live bacterial cells

    CERN Document Server

    Deng, Yi; Shaevitz, Joshua W

    2011-01-01

    The mechanical properties of gram-negative bacteria are governed by a rigid peptidoglycan (PG) cell wall and the turgor pressure generated by the large concentration of solutes in the cytoplasm. The elasticity of the PG has been measured in bulk and in isolated sacculi and shown to be compliant compared to the overall stiffness of the cell itself. However, the stiffness of the cell wall in live cells has not been measured. In particular, the effects that pressure-induced stress might have on the stiffness of the mesh-like PG network have not been addressed even though polymeric materials often exhibit large amounts of stress-stiffening. We study bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of $1.07 \\pm 0.25$, such that the wall is significantly stiffer in live cells ($E\\sim32\\pm10$ MPa) than in unpres...

  20. Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Seonghee Lee

    2013-02-01

    Full Text Available Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR, which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance.

  1. Single-cell level based approach to investigate bacterial metabolism during batch industrial fermentation

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.;

    , and performance of Escherichia coli. An insight into glucose and acetate fate on the level of individual cell can provide the type of information which are valuable for the understanding of bacterial metabolism in fermentation process and can shed more light on the differentiation of isogenic fermenting...... can exhibit different phenotypes under specific environmental conditions that show significant differences in physiological parameters from the population average. However, studies concerning segregation of populations into metabolically diversified subpopulations are scarce. Acetate is a product...... of Escherichia coli overflow metabolism when the bacteria are grown under aerobic conditions and glucose is present in excessive concentrations. Acetate accumulation is of the utmost importance in batch fermentation processes as it is an undesirable byproduct that negatively affects growth, physiology...

  2. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  3. Bacterial nanocellulose/Nafion composite membranes for low temperature polymer electrolyte fuel cells

    Science.gov (United States)

    Jiang, Gao-peng; Zhang, Jing; Qiao, Jin-li; Jiang, Yong-ming; Zarrin, Hadis; Chen, Zhongwei; Hong, Feng

    2015-01-01

    Novel nanocomposite membranes aimed for both proton-exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) are presented in this work. The membranes are based on blending bacterial nanocellulose pulp and Nafion (abbreviated as BxNy, where x and y indicates the mass ratio of bacterial cellulose to Nafion). The structure and properties of BxNy membranes are characterized by FTIR, SEM, TG, DMA and EIS, along with water uptake, swelling behavior and methanol permeability tests. It is found that the BxNy composite membranes with reinforced concrete-like structure show excellent mechanical and thermal stability regardless of annealing. The water uptake plus area and volume swelling ratios are all decreased compared to Nafion membranes. The proton conductivities of pristine and annealed B1N9 are 0.071 and 0.056 S cm-1, respectively, at 30 °C and 100% humidity. Specifically, annealed B1N1 exhibited the lowest methanol permeability of 7.21 × 10-7 cm2 s-1. Through the selectivity analysis, pristine and annealed B1N7 are selected to assemble the MEAs. The performances of annealed B1N7 in PEMFC and DMFC show the maximum power densities of 106 and 3.2 mW cm-2, respectively, which are much higher than those of pristine B1N7 at 25 °C. The performances of the pristine and annealed B1N7 reach a level as high as 21.1 and 20.4 mW cm-2 at 80 °C in DMFC, respectively.

  4. Phenotypic T cell exhaustion in a murine model of bacterial infection in the setting of pre-existing malignancy.

    Science.gov (United States)

    Mittal, Rohit; Wagener, Maylene; Breed, Elise R; Liang, Zhe; Yoseph, Benyam P; Burd, Eileen M; Farris, Alton B; Coopersmith, Craig M; Ford, Mandy L

    2014-01-01

    While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8+ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8+ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8+ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8+ T cells and total CD4+ and CD8+ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8+ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8+ T cell functionality and differentiation. PMID:24796533

  5. Phenotypic T cell exhaustion in a murine model of bacterial infection in the setting of pre-existing malignancy.

    Directory of Open Access Journals (Sweden)

    Rohit Mittal

    Full Text Available While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8+ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8+ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8+ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8+ T cells and total CD4+ and CD8+ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8+ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8+ T cell functionality and differentiation.

  6. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  7. Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

    Science.gov (United States)

    Manti, Anita; Boi, Paola; Amalfitano, Stefano; Puddu, Alberto; Papa, Stefano

    2011-12-01

    Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

  8. Sensing the Structural Differences in Cellulose from Apple and Bacterial Cell Wall Materials by Raman and FT-IR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Artur Zdunek

    2011-05-01

    Full Text Available Raman and Fourier Transform Infrared (FT-IR spectroscopy was used for assessment of structural differences of celluloses of various origins. Investigated celluloses were: bacterial celluloses cultured in presence of pectin and/or xyloglucan, as well as commercial celluloses and cellulose extracted from apple parenchyma. FT-IR spectra were used to estimate of the Iβ content, whereas Raman spectra were used to evaluate the degree of crystallinity of the cellulose. The crystallinity index (XCRAMAN% varied from −25% for apple cellulose to 53% for microcrystalline commercial cellulose. Considering bacterial cellulose, addition of xyloglucan has an impact on the percentage content of cellulose Iβ. However, addition of only xyloglucan or only pectins to pure bacterial cellulose both resulted in a slight decrease of crystallinity. However, culturing bacterial cellulose in the presence of mixtures of xyloglucan and pectins results in an increase of crystallinity. The results confirmed that the higher degree of crystallinity, the broader the peak around 913 cm−1. Among all bacterial celluloses the bacterial cellulose cultured in presence of xyloglucan and pectin (BCPX has the most similar structure to those observed in natural primary cell walls.

  9. Ascites bacterial burden and immune cell profile are associated with poor clinical outcomes in the absence of overt infection.

    Directory of Open Access Journals (Sweden)

    Kevin J Fagan

    Full Text Available Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non-neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission. Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both.

  10. Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Dalton, Helen M.; Angels, Mark;

    1997-01-01

    A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells: a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of Fluorescently labelled oligonucleotide probes to rRNA. The method a...

  11. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    Energy Technology Data Exchange (ETDEWEB)

    Economou, Nicoleta J.; Zentner, Isaac J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States); Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian [Brookhaven National Laboratory, Upton, NY 11973 (United States); Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States)

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  12. BACTERIAL BIOFILM FORMATION VERSUS MAMMALIAN CELL GROWTH ON TITANIUM-BASED MONO- AND BI-FUNCTIONAL COATINGS

    NARCIS (Netherlands)

    Subbiahdoss, Guruprakash; Pidhatika, Bidhari; Coullerez, Geraldine; Charnley, Mirren; Kuijer, Roel; van der Mei, Henny C.; Textor, Marcus; Busscher, Henk J.

    2010-01-01

    Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while s

  13. The contribution of cell-cell signaling and motility to bacterial biofilm formation

    DEFF Research Database (Denmark)

    Shrout, Joshua D; Tolker-Nielsen, Tim; Givskov, Michael;

    2011-01-01

    Many bacteria grow attached to a surface as biofilms. Several factors dictate biofilm formation, including responses by the colonizing bacteria to their environment. Here we review how bacteria use cell-cell signaling (also called quorum sensing) and motility during biofilm formation. Specifically...... gene expression important to the production of polysaccharides, rhamnolipid, and other virulence factors. Surface motility affects the assembly and architecture of biofilms, and some aspects of motility are also influenced by quorum sensing. While some genes and their function are specific to P....... aeruginosa, many aspects of biofilm development can be used as a model system to understand how bacteria differentially colonize surfaces....

  14. X-ray crystallography and its impact on understanding bacterial cell wall remodeling processes.

    Science.gov (United States)

    Büttner, Felix Michael; Renner-Schneck, Michaela; Stehle, Thilo

    2015-02-01

    The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding.

  15. Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

    Directory of Open Access Journals (Sweden)

    Tran Ngat

    2011-05-01

    Full Text Available Abstract Background A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2 as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin that target distinct stages of cell wall biosynthesis. Results A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σE, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σB or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. Conclusions Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional.

  16. Modeling nucleotide excision repair and its impact on UV-induced mutagenesis during SOS-response in bacterial cells.

    Science.gov (United States)

    Bugay, Aleksandr N; Krasavin, Evgeny A; Parkhomenko, Aleksandr Yu; Vasilyeva, Maria A

    2015-01-01

    A model of the UV-induced mutation process in Escherichia coli bacteria has been developed taking into account the whole sequence of molecular events starting from initial photo-damage and finishing with the fixation of point mutations. The wild-type phenotype bacterial cells are compared with UV-sensitive repair-deficient mutant cells. Attention is mainly paid to excision repair system functioning as regards induced mutagenesis.

  17. Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

    Directory of Open Access Journals (Sweden)

    Grégory Francius

    Full Text Available The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM and electrokinetics (electrophoresis. Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus. From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700-900 kPa and ∼100-300 kPa respectively. Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the

  18. Modified bacterial cellulose scaffolds for localized doxorubicin release in human colorectal HT-29 cells.

    Science.gov (United States)

    Cacicedo, Maximiliano L; León, Ignacio E; Gonzalez, Jimena S; Porto, Luismar M; Alvarez, Vera A; Castro, Guillermo R

    2016-04-01

    Bacterial cellulose (BC) films modified by the in situ method with the addition of alginate (Alg) during the microbial cultivation of Gluconacetobacter hansenii under static conditions increased the loading of doxorubicin by at least three times. Biophysical analysis of BC-Alg films by scanning electron microscopy, thermogravimetry, X-ray diffraction and FTIR showed a highly homogeneous interpenetrated network scaffold without changes in the BC crystalline structure but with an increased amorphous phase. The main molecular interactions determined by FTIR between both biopolymers clearly suggest high compatibility. These results indicate that alginate plays a key role in the biophysical properties of the hybrid BC matrix. BC-Alg scaffold analysis by nitrogen adsorption isotherms revealed by the Brunauer-Emmett-Teller (BET) method an increase in surface area of about 84% and in pore volume of more than 200%. The Barrett-Joyner-Halenda (BJH) model also showed an increase of about 25% in the pore size compared to the BC film. Loading BC-Alg scaffolds with different amounts of doxorubicin decreased the cell viability of HT-29 human colorectal adenocarcinoma cell line compared to the free Dox from around 95-53% after 24h and from 63% to 37% after 48 h. Dox kinetic release from the BC-Alg nanocomposite displayed hyperbolic curves related to the different amounts of drug payload and was stable for at least 14 days. The results of the BC-Alg nanocomposites show a promissory potential for anticancer therapies of solid tumors.

  19. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2016-08-01

    Full Text Available Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs, have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated proteins in bacterial cells and the perspectives on how to overcome the issues.

  20. A 16 × 16 CMOS Capacitive Biosensor Array Towards Detection of Single Bacterial Cell.

    Science.gov (United States)

    Couniot, Numa; Francis, Laurent A; Flandre, Denis

    2016-04-01

    We present a 16 × 16 CMOS biosensor array aiming at impedance detection of whole-cell bacteria. Each 14 μm × 16 μm pixel comprises high-sensitive passivated microelectrodes connected to an innovative readout interface based on charge sharing principle for capacitance-to-voltage conversion and subthreshold gain stage to boost the sensitivity. Fabricated in a 0.25 μm CMOS process, the capacitive array was experimentally shown to perform accurate dielectric measurements of the electrolyte up to electrical conductivities of 0.05 S/m, with maximal sensitivity of 55 mV/fF and signal-to-noise ratio (SNR) of 37 dB. As biosensing proof of concept, real-time detection of Staphylococcus epidermidis binding events was experimentally demonstrated and provides detection limit of ca. 7 bacteria per pixel and sensitivity of 2.18 mV per bacterial cell. Models and simulations show good matching with experimental results and provide a comprehensive analysis of the sensor and circuit system. Advantages, challenges and limits of the proposed capacitive biosensor array are finally described with regards to literature. With its small area and low power consumption, the present capacitive array is particularly suitable for portable point-of-care (PoC) diagnosis tools and lab-on-chip (LoC) systems. PMID:25974947

  1. Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections.

    Science.gov (United States)

    Pashenkov, Mikhail; Teleshova, Natalia; Kouwenhoven, Mathilde; Smirnova, Tatiana; Jin, Ya Ping; Kostulas, Vasilios; Huang, Yu Min; Pinegin, Boris; Boiko, Alexey; Link, Hans

    2002-01-01

    Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c(+)CD123(dim)) and plasmacytoid (CD11c(-)CD123(high)) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1alpha were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1alpha, and CD88, the receptor for C5a. SDF-1alpha present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

  2. The impact of hypoxia on intestinal epithelial cell functions: consequences for invasion by bacterial pathogens.

    Science.gov (United States)

    Zeitouni, Nathalie E; Chotikatum, Sucheera; von Köckritz-Blickwede, Maren; Naim, Hassan Y

    2016-12-01

    The maintenance of oxygen homeostasis in human tissues is mediated by several cellular adaptations in response to low-oxygen stress, called hypoxia. A decrease in tissue oxygen levels is initially counteracted by increasing local blood flow to overcome diminished oxygenation and avoid hypoxic stress. However, studies have shown that the physiological oxygen concentrations in several tissues are much lower than atmospheric (normoxic) conditions, and the oxygen supply is finely regulated in individual cell types. The gastrointestinal tract has been described to subsist in a state of physiologically low oxygen level and is thus depicted as a tissue in the state of constant low-grade inflammation. The intestinal epithelial cell layer plays a vital role in the immune response to inflammation and infections that occur within the intestinal tissue and is involved in many of the adaptation responses to hypoxic stress. This is especially relevant in the context of inflammatory disorders, such as inflammatory bowel disease (IBD). Therefore, this review aims to describe the intestinal epithelial cellular response to hypoxia and the consequences for host interactions with invading gastrointestinal bacterial pathogens. PMID:27002817

  3. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets.

    Science.gov (United States)

    Liu, Yao; Breukink, Eefjan

    2016-01-01

    Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs), have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated) proteins in bacterial cells and the perspectives on how to overcome the issues. PMID:27571111

  4. Modified bacterial cellulose scaffolds for localized doxorubicin release in human colorectal HT-29 cells.

    Science.gov (United States)

    Cacicedo, Maximiliano L; León, Ignacio E; Gonzalez, Jimena S; Porto, Luismar M; Alvarez, Vera A; Castro, Guillermo R

    2016-04-01

    Bacterial cellulose (BC) films modified by the in situ method with the addition of alginate (Alg) during the microbial cultivation of Gluconacetobacter hansenii under static conditions increased the loading of doxorubicin by at least three times. Biophysical analysis of BC-Alg films by scanning electron microscopy, thermogravimetry, X-ray diffraction and FTIR showed a highly homogeneous interpenetrated network scaffold without changes in the BC crystalline structure but with an increased amorphous phase. The main molecular interactions determined by FTIR between both biopolymers clearly suggest high compatibility. These results indicate that alginate plays a key role in the biophysical properties of the hybrid BC matrix. BC-Alg scaffold analysis by nitrogen adsorption isotherms revealed by the Brunauer-Emmett-Teller (BET) method an increase in surface area of about 84% and in pore volume of more than 200%. The Barrett-Joyner-Halenda (BJH) model also showed an increase of about 25% in the pore size compared to the BC film. Loading BC-Alg scaffolds with different amounts of doxorubicin decreased the cell viability of HT-29 human colorectal adenocarcinoma cell line compared to the free Dox from around 95-53% after 24h and from 63% to 37% after 48 h. Dox kinetic release from the BC-Alg nanocomposite displayed hyperbolic curves related to the different amounts of drug payload and was stable for at least 14 days. The results of the BC-Alg nanocomposites show a promissory potential for anticancer therapies of solid tumors. PMID:26784658

  5. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

    Science.gov (United States)

    Belcourt, M F; Penketh, P G; Hodnick, W F; Johnson, D A; Sherman, D H; Rockwell, S; Sartorelli, A C

    1999-08-31

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  6. Biosynthesis of Bacterial Cellulose/Carboxylic Multi-Walled Carbon Nanotubes for Enzymatic Biofuel Cell Application

    Directory of Open Access Journals (Sweden)

    Pengfei Lv

    2016-03-01

    Full Text Available Novel nanocomposites comprised of bacterial cellulose (BC with carboxylic multi-walled carbon nanotubes (c-MWCNTs incorporated into the BC matrix were prepared through a simple method of biosynthesis. The biocathode and bioanode for the enzyme biological fuel cell (EBFC were prepared using BC/c-MWCNTs composite injected by laccase (Lac and glucose oxidase (GOD with the aid of glutaraldehyde (GA crosslinking. Biosynthesis of BC/c-MWCNTs composite was characterized by digital photos, scanning electron microscope (SEM, and Fourier Transform Infrared (FTIR. The experimental results indicated the successful incorporation of c-MWCNTs into the BC. The electrochemical and biofuel performance were evaluated by cyclic voltammetry (CV and linear sweep voltammetry (LSV. The power density and current density of EBFCs were recorded at 32.98 µW/cm3 and 0.29 mA/cm3, respectively. Additionally, the EBFCs also showed acceptable stability. Preliminary tests on double cells indicated that renewable BC have great potential in the application field of EBFCs.

  7. Structure of Ristocetin A in Complex with a Bacterial Cell-wall Mimetic

    Energy Technology Data Exchange (ETDEWEB)

    Nahoum, V.; Spector, S; Loll, P

    2009-01-01

    Antimicrobial drug resistance is a serious public health problem and the development of new antibiotics has become an important priority. Ristocetin A is a class III glycopeptide antibiotic that is used in the diagnosis of von Willebrand disease and which has served as a lead compound for the development of new antimicrobial therapeutics. The 1.0 A resolution crystal structure of the complex between ristocetin A and a bacterial cell-wall peptide has been determined. As is observed for most other glycopeptide antibiotics, it is shown that ristocetin A forms a back-to-back dimer containing concave binding pockets that recognize the cell-wall peptide. A comparison of the structure of ristocetin A with those of class I glycopeptide antibiotics such as vancomycin and balhimycin identifies differences in the details of dimerization and ligand binding. The structure of the ligand-binding site reveals a likely explanation for ristocetin A's unique anticooperativity between dimerization and ligand binding.

  8. Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

    Directory of Open Access Journals (Sweden)

    Carlson Petteri

    2010-10-01

    Full Text Available Abstract Background Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD, which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. Results Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. Conclusions These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

  9. Relationship among specific bacterial counts and total bacterial and somatic cell counts and factors influencing their variation in ovine bulk tank milk.

    Science.gov (United States)

    de Garnica, M L; Linage, B; Carriedo, J A; De La Fuente, L F; García-Jimeno, M C; Santos, J A; Gonzalo, C

    2013-02-01

    To analyze the relationship among the counts of different organisms and total bacterial count (BTTBC) and somatic cell count (BTSCC) as determined in dairy laboratories in ovine bulk tank milk, 751 bulk tank milk samples from 205 dairy sheep flocks belonging to Consortium for Ovine Promotion (CPO) were collected between January and December 2011. Four samplings were carried out in each flock, once per season, throughout 1 yr. Variables analyzed were bulk tank counts of thermoduric, psychrotrophic, coliform, and gram-positive catalase-negative cocci (GPCNC) bacterial groups. Thermoduric, psychrotrophic, and coliform species were significantly related to BTTBC, whereas GPCNC were correlated with both BTTBC and BTSCC variables. Highest counts were for psychrotroph and coliform groups, and a moderate to high correlation (r=0.51) was found between both variables, indicating that poor cleaning practices in the flocks tend to select for less-resistant organisms, such as gram-negative rods. In addition, BTTBC correlated with BTSCC (r=0.42). Some variation factors for specific bacterial counts, such as breed, season, milking type, dry therapy, and milk yield, were also analyzed. Flock information was collected from flock books, annual audits, and the CPO traceability system. Psychrotrophs and coliforms had elevated counts in winter, whereas GPCNC were higher in summer and in hand-milked flocks. Dry therapy contributed to the reduction in psychrotrophic bacteria; therefore, some strains of mammary pathogens could also be psychrotrophic bacteria. Results of this study would be helpful for troubleshooting milk quality problems and developing premium payment systems in dairy sheep.

  10. Vigorous, but differential mononuclear cell response of cirrhotic patients to bacterial ligands

    Institute of Scientific and Technical Information of China (English)

    Varenka J Barbero-Becerra; María Concepción Gutiérrez-Ruiz; Carmen Maldonado-Bernal; Félix I Téllez-Avila; Roberto Alfaro-Lara; Florencia Vargas-Vorácková

    2011-01-01

    AIM: To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS: Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis. Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor (TNF) α, interleukin (IL)-1β, IL-6, IL-12 and IL-10 secretion in supernatants was determined by ELISA. RESULTS: Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononuclear cells from cirrhotic patients, (P < 0.01, P < 0.006, P < 0.111) respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression (P < 0.111 lipopolysaccharide, P < 0.013 lipoteichoic acid), and a decrease in Toll-like receptor 2 (P < 0.008 lipopolysaccharide, P < 0.008 lipoteichoic acid) and Toll-like receptor 4 (P < 0.008 lipopolysaccharide, P < 0.028 lipoteichoic acid) expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions (P < 0.070 IL-6, P < 0.009 IL-1β, P < 0.022 IL-12). Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former (P < 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P < 0.037 TNFα; P < 0.006 IL-1β; P < 0.005 IL-6; P < 0.007 IL-12; P < 0.014 IL-10 lipoteichoic acid). Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION: Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide

  11. Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

    Science.gov (United States)

    Hayward, Richard D; Cain, Robert J; McGhie, Emma J; Phillips, Neil; Garner, Matthew J; Koronakis, Vassilis

    2005-05-01

    A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

  12. Studies on interfacial interactions of TiO2 nanoparticles with bacterial cells under light and dark conditions

    Indian Academy of Sciences (India)

    Swayamprava Dalai; Sunandan Pakrashi; Sujay Chakravarty; Shamima Hussain; N Chandrasekaran; Amitava Mukherjee

    2014-05-01

    The probable underlying mechanism(s) of bacterial cell–TiO2 nanoparticles (TiO2 NPs) interaction in the absence of photo-irradiation has been less studied since most of the prior cytotoxicity studies focused on irradiated TiO2. The present study draws attention to the possible role of cell surface–TiO2 NP interactions under dark conditions, through an array of spectroscopic and microscopic investigations. A dominant freshwater bacterial isolate, Bacillus licheniformis, which interacted with environmentally relevant concentrations of TiO2 NPs (1 g/mL), was analysed and compared under both light and dark conditions. Aggregation of cells upon NP interaction and adsorption of NPs onto the cell membrane was evident from the scanning electron micrographs under both light and dark conditions. The FT–IR and FT–Raman spectra suggested stress response of bacterial cells by elevated protein and polysaccharide content in the cell–NP interaction. The X-ray photoelectron spectroscopic data substantiated the reduction of titanium from Ti(IV) to Ti(III) species which might have contributed to the redox interactions on the cell surface under light as well as dark conditions. The internalization of NPs in the cytoplasm were obvious from the transmission electron micrographs. The consequent cell death/damage was confirmed through fluorescence spectroscopy and microscopy. To conclude, the current study established the substantial role of interfacial interactions in cytotoxicity of the TiO2 NPs irrespective of the irradiation conditions.

  13. Activation of p38α in T cells regulates the intestinal host defense against attaching and effacing bacterial infections

    OpenAIRE

    Shim, Eun-Jin; Bang, Bo Ram; Kang, Seung-Goo; Ma, Jianhui; Otsuka, Motoyuki; Kang, Jiman; Stahl, Martin; Han, Jiahuai; Xiao, Changchun; Vallance, Bruce A.; Kang, Young Jun

    2013-01-01

    Intestinal infections by attaching and effacing (A/E) bacterial pathogens cause severe colitis and bloody diarrhea. Although p38α in intestine epithelial cells (IEC) plays an important role in promoting protection against A/E bacteria by regulating T cell recruitment, its impact on immune responses remains unclear. In this study, we show that activation of p38α in T cells is critical for the clearance of the A/E pathogen Citrobacter rodentium. Mice deficient of p38α in T cells, but not in mac...

  14. Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.

    Science.gov (United States)

    Hajduk, Isabella V; Rodrigues, Christopher D A; Harry, Elizabeth J

    2016-05-01

    Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells.

  15. Bacterial β-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells.

    Science.gov (United States)

    Lee, Kwang-Ho; Park, Min; Ji, Kon-Young; Lee, Hwa-Youn; Jang, Ji-Hun; Yoon, Il-Joo; Oh, Seung-Su; Kim, Su-Man; Jeong, Yun-Hwa; Yun, Chul-Ho; Kim, Mi-Kyoung; Lee, In-Young; Choi, Ha-Rim; Ko, Ki-sung; Kang, Hyung-Sik

    2014-10-01

    Bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial β-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial β-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial β-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and IL-17A/F, were markedly decreased in the colon of β-(1,3)-glucan-pretreated mice. β-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, β-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial β-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

  16. In vitro behaviors of rat mesenchymal stem cells on bacterial celluloses with different moduli

    Energy Technology Data Exchange (ETDEWEB)

    Taokaew, Siriporn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States); Phisalaphong, Muenduen [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Zhang Newby, Bi-min, E-mail: bimin@uakron.edu [Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States)

    2014-05-01

    Compressive moduli of bacteria-synthesized cellulose (BC) were altered by two drying techniques: ambient-air drying and freeze drying. While no significant differences in dry weight were found, their cross-sectional structures and thickness varied greatly. Freeze dried BCs had loose cross-sectional structures and a thickness of ∼ 4.7 mm, whereas air dried BCs had more compacted cross-sectional structures and a thickness of ∼ 0.1 mm. The compressive moduli of the rehydrated freeze dried and rehydrated air dried BCs were measured to be 21.06 ± 0.22 kPa and 90.09 ± 21.07 kPa, respectively. When rat mesenchymal stem cells (rMSCs) were seeded on these BCs, they maintained a round morphology in the first 3 days of cultivation. More spread-out morphology and considerable proliferation on freeze dried BCs were observed in 7 days, but not on air-dried BCs. The cells were further grown for 3 weeks in the absence and presence of differentiation agents. Without using any differentiation agents, no detectable differentiation was noticed for rMSCs further cultivated on both types of BC. With differentiation inducing agents, chondrogenic differentiation, visualized by histological staining, was observed in some area of the rehydrated freeze dried BCs; while osteogenic differentiation was noticed on the stiffer rehydrated air dried BCs. - Graphical abstract: In the presence of induction agents, rat mesenchymal stem cells (rMSCs) preferentially differentiated into osteocytes on stiffer air dried BC films. - Highlights: • Bacterial cellulose (BC) sheets with different moduli generated by drying differently • Air-dried BC exhibited a modulus similar to that of bone. • Freeze-dried BC showed a modulus in the range of that of muscle. • Air-dried BC promoted the differentiation of rMSCs into osteocytes. • Freeze-dried BC promoted the differentiation of rMSCs into chondrocytes.

  17. Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

    Science.gov (United States)

    Tixador, R.

    1992-01-01

    The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

  18. Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

    DEFF Research Database (Denmark)

    Kaartokallio, H.; Søgaard, D.H.; Norman, L.;

    2013-01-01

    and cell population properties (by flow cytometry) in subarctic sea ice in SW Greenland. Short-term temporal variability was moderate, and steep environmental gradients, typical for sea ice, were the main drivers of the variability in bacterial cell properties and activity. Low nucleic acid (LNA) bacteria......Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance......, previously linked to oligotrophic ecotypes in marine habitats, were more abundant in the upper ice layers, whereas high nucleic acid (HNA) bacteria dominated in lower ice, where organic carbon was in high concentrations. Leu incorporation was saturated at micromolar concentrations, as known from freshwater...

  19. Reinforcement of epithelial cell adhesion to basement membrane by a bacterial pathogen as a new infectious stratagem.

    Science.gov (United States)

    Kim, Minsoo; Ogawa, Michinaga; Mimuro, Hitomi; Sasakawa, Chihiro

    2010-01-01

    The intestinal epithelium undergoes a rapid turnover in addition to rapid exfoliation in response to bacterial infection, thus acting as an intrinsic defense against microbial intruders. It has long been questioned how mucosal pathogens can circumvent the intestinal defense systems. Our recent discovery of a bacterial ploy used by Shigella provided us with fresh insight. Shigella delivers OspE via the type III secretion system during multiplication within epithelial cells. This effector protein reinforces epithelial adherence to the basement membrane by interacting with integrin-linked kinase (ILK), a unique intracellular Ser/Thr kinase that links the cell-adhesion receptors, integrin, and growth factors to the actin cytoskeleton. The interaction between OspE and ILK increased formation of focal adhesions (FAs) and surface levels of b1-integrin, while suppressing phosphorylation of FAK and paxillin, thus suppressing rapid turnover of FAs, reducing cell motility and promoting cell adhesion to extracellular matrix. The impact of this OspE-ILK interplay was demonstrated both in vitro and in vivo by infecting polarized epithelial cell monolayers and guinea pig colons with Shigella possessing or lacking the ospE gene. The findings thus establish a new class of virulence-associated factors, and provide new insight into the functioning of the intestinal barrier and bacterial strategies for circumventing it. PMID:21178415

  20. A novel functional T cell hybridoma recognizes macrophage cell death induced by bacteria: a possible role for innate lymphocytes in bacterial infection.

    Science.gov (United States)

    Kubota, Koichi

    2006-06-15

    We have established a novel TCRalphabeta (TCRVbeta6)(+)CD4(-)CD8(-) T cell hybridoma designated B6HO3. When the B6HO3 cells were cocultured with bacterial-infected J774 macrophage-like cells, IFN-gamma production by B6HO3 cells was triggered through direct cell-cell contact with dying J774 cells infected with Listeria monocytogenes (LM), Shigella flexneri, or Salmonella typhimurium that expressed the type III secretion system, but not with intact J774 cells infected with heat-killed LM, nonhemolytic lysteriolysin O-deficient (Hly(-)) LM, plasmid-cured Shigella, or stationary-phase Salmonella. However, the triggering of B6HO3 cells for IFN-gamma production involved neither dying hepatoma cells infected with LM nor dying J774 cells caused by gliotoxin treatment or freeze thawing. Cycloheximide and Abs to H-2K(d), H-2D(d), Ia(d), CD1d, TCRVbeta6, and IL-12 did not inhibit the contact-dependent IFN-gamma response, indicating that this IFN-gamma response did not require de novo protein synthesis in bacterial-infected J774 cells and was TCR and IL-12 independent. Thus, in an as yet undefined way, B6HO3 hybridoma recognizes a specialized form of macrophage cell death resulting from bacterial infection and consequently produces IFN-gamma. Moreover, contact-dependent interaction of minor subsets of splenic alphabeta T cells, including NKT cells with dying LM-infected J774 and bone marrow-derived macrophage (BMM) cells, proved to provide an IFN-gamma-productive stimulus for these minor T cell populations, to which the parental T cell of the B6HO3 hybridoma appeared to belong. Unexpectedly, subsets of gammadelta T and NK cells similarly responded to dying LM-infected macrophage cells. These results propose that innate lymphocytes may possess a recognition system sensing macrophage cell "danger" resulting from bacterial infection. PMID:16751404

  1. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  2. An ethanol biosensor based on a bacterial cell-immobilized eggshell membrane

    Institute of Scientific and Technical Information of China (English)

    Guang Ming Wen; Shao Min Shuang; Chuan Dong; Martin M.F. Choi

    2012-01-01

    An ethanol biosensor was fabricated based on a Methylobacterium organophilium-immobilized eggshell membrane and an oxygen (O2) electrode.A linear response for ethanol was obtained in the range of 0.050-7.5 mmol/L with a detection limit of 0.025 mmol/L (S/N =3) and a R.S.D.of 2.1%.The response time was less than 100 s at room temperature and ambient pressure.The optimal loading of bacterial cells on the biosensor membrane is 40 mg (wet weight).The optimal working conditions for the microbial biosensor are pH 7.0 phosphate buffer (50 mmol/L) at 20-25 ℃.The interference test,operational and storage stability of the biosensor are studied in detail.Finally,the biosensor is applied to determine the ethanol contents in various alcohol samples and the results are comparable to that obtained by gas chromatographic method and the results are satisfactory.Our proposed biosensor provides a convenient,simple and reliable method to determine ethanol content in alcoholic drinks.

  3. Interaction of Gram-negative bacteria with cationic proteins: Dependence on the surface characteristics of the bacterial cell

    Directory of Open Access Journals (Sweden)

    Isabella R Prokhorenko

    2009-03-01

    Full Text Available Isabella R Prokhorenko1, Svetlana V Zubova1, Alexandr Yu Ivanov2, Sergey V Grachev31Laboratory of Molecular Biomedicine, Institute of Basic Biological Problems; 2Institute of Cell Biophysics, Russian Academy of Sciences, Moscow, Russia; 3I.M. Sechenov’s Moscow Medical Academy, Moscow, Russia Abstract: Gram-negative bacteria can enter the bloodstream and interact with serum cationic proteins. The character of interaction will depend on the surface characteristics of bacterial cells, which are determined by bacterial chemotype and density of lipopolysaccharide (LPS packing in the cell wall. It was shown that the lysozyme treatment resulted in the increase sensitivity to hypotonic shock. Signifi cant differences to this effect were found between Escherichia coli strain D21 and D21f2 under treatment with physiological protein concentration. On the basis of electrokinetic measurements and studies of the interaction of cells with lysozyme, the hypothesis was formed that the cell wall of the E. coli strain D21f2 contains more LPS and has a higher density of their packing than the cell wall of the E. coli D21 cells. The effect of lysozyme and lactoferrin on the viability of E. coli cells of two different strains was examined. Lysozyme was found to more effectively inhibit the growth of the E. coli D21 bacteria, and lactoferrin suppressed mainly the growth of the E. coli D21f2 bacteria. These results indicate that the differences in LPS core structure of bacterial R-chemotype, which determines surface charge and density of LPS packing, plays an essential role in the mechanisms of interaction of the cationic proteins with the cell wall.Keywords: lipopolysaccharide, Escherichia coli, chemotype, lysozyme, lactoferrin, colony-forming units

  4. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

    Directory of Open Access Journals (Sweden)

    Katrin Bomans

    Full Text Available Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS. However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day manner.

  5. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  6. Bacterial CD1d-restricted glycolipids induce IL-10 production by human regulatory T cells upon cross-talk with invariant NKT cells.

    Science.gov (United States)

    Venken, Koen; Decruy, Tine; Aspeslagh, Sandrine; Van Calenbergh, Serge; Lambrecht, Bart N; Elewaut, Dirk

    2013-09-01

    Invariant NKT (iNKT) cells and CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell-based therapeutics.

  7. On setting the first dose in man: quantitating biotherapeutic drug-target binding through pharmacokinetic and pharmacodynamic models.

    Science.gov (United States)

    Lowe, Philip J; Tannenbaum, Stacey; Wu, Kai; Lloyd, Peter; Sims, Jennifer

    2010-03-01

    Although the three (perhaps four) phases of clinical drug development are well known, it is relatively unappreciated that there are similar phases in pre-clinical development. These consist of 'Phase I' the initial, normally Research Discovery driven pharmacology; 'Phase II' non-good laboratory practice (GLP) dose range finding, followed by pivotal 'Phase III' GLP toxicology. Together with an array of in vitro experiments comparing species, these stages should enable an integrated safety assessment prior to entry into man, documenting to investigators and authorities evidence that the new pharmaceutic is unlikely to cause harm. Following the lessons learned from TeGenero TGN1412 and subsequent updates to regulatory guidelines, there are aspects peculiar to biotherapeutics, especially those that target key body systems, where calculations could be made for doses for human studies using pharmacokinetic and pharmacodynamic models. Two of these are exemplified in this paper. In the first, target-mediated drug disposition, where the binding of the drug to a cellular target quantitatively affects the pharmacokinetics, enables occupancy to be estimated without recourse to independent assays. In the second, assaying captured soluble target, as drug-target complexes, allows estimation of the concentration of the free ligand ensuring that in initial clinical studies, soluble targets are not overly suppressed. To support this methodology, it has been demonstrated using omalizumab, free and total IgE data that such analyses do predict the suppression of the free unbound ligand with reasonable accuracy. Overall, the objective of the process is to deliver a justification, through consideration of drug-target binding, of a safe starting and therapeutically relevant escalation doses for human studies. PMID:20050847

  8. Potencial bioterapêutico dos probióticos nas parasitoses intestinais Probiotics as potential biotherapeutic agents targeting intestinal parasites

    Directory of Open Access Journals (Sweden)

    Teresa Cristina Goulart de Oliveira-Sequeira

    2008-12-01

    Full Text Available Probióticos são microrganismos vivos que, se administrados em quantidades adequadas, promovem benefícios à saúde do homem e dos animais. O crescente interesse nos probióticos fundamenta-se em estudos clínicos nos quais a administração desses organismos foi avaliada na prevenção e no tratamento de desordens intestinais e sistêmicas. Os potenciais mecanismos de ação desses microrganismos incluem a exclusão competitiva, a produção de metabólitos com atividade antimicrobiana e a modulação da resposta imune. Em algumas circunstâncias clínicas específicas, os benefícios produzidos por esses microrganismos foram amplamente documentados, enquanto que em outras os resultados são contraditórios. No presente artigo de revisão, os probióticos foram abordados considerando-se o potencial bioterapêutico desses microrganismos nas parasitoses intestinais.Probiotics are live microorganisms which, when administered in adequate amounts, beneficially affect the general health status of man and animal. The great interest in probiotic microganisms is based on evidences from clinical studies indicating benefits in the prevention or treatment of a broad spectrum of gastrointestinal and systemic disorders. The potential mechanisms by which probiotics beneficially affect health include strengthening of the intestinal barrier, modulation of the immune response, and antagonism of pathogens either by the production of antimicrobial compounds or through competition for mucosal binding sites. In some specific clinical circumstances, there is clear evidence of benefit whereas in others, the results are dubious and important questions remaining unanswered. The aim of this review article is to focus probiotics on their potential as biotherapeutic agents against intestinal parasites.

  9. Effect of bacterial lectin on acceleration of fat cell lipolysis at in vitro diode laser treatment using encapsulated ICG

    Science.gov (United States)

    Yanina, Irina Yu.; Kochubey, Vyacheslav I.; Tuchin, Valery V.; Portnov, Sergey A.; Svenskaya, Yuliya I.; Gorin, Dmitry A.; Ponomareva, Elena G.; Nikitina, Valentina E.

    2012-03-01

    The influence of bacterial lectin on photochemically induced fat cell lipolysis was studied. Resulting capsules were tested for ICG absorption by optical spectra measurements. To separate released and encapsulated ICG supernatant was removed and capsules were redispered in pure deionized water. Supernatant and capsule suspension spectra were measured separately. It was also found that pretreatment of tissue by lectin leads to acceleration of lipolysis at photochemical treatment. The data obtained can be used to enhance efficiency of photochemical therapy.

  10. Effects of season, milking routine and cow cleanliness on bacterial and somatic cell counts of bulk tank milk.

    Science.gov (United States)

    Zucali, Maddalena; Bava, Luciana; Tamburini, Alberto; Brasca, Milena; Vanoni, Laura; Sandrucci, Anna

    2011-11-01

    The aim of the study was to investigate the effects of season, cow cleanliness and milking routine on bacterial and somatic cell counts of bulk tank milk. A total of 22 dairy farms in Lombardy (Italy) were visited three times in a year in different seasons. During each visit, samples of bulk tank milk were taken for bacterial and somatic cell counts; swabs from the teat surface of a group of cows were collected after teat cleaning and before milking. Cow cleanliness was assessed by scoring udder, flanks and legs of all milking cows using a 4-point scale system. Season affected cow cleanliness with a significantly higher percentage of non-clean (NC) cows during Cold compared with Mild season. Standard plate count (SPC), laboratory pasteurization count (LPC), coliform count (CC) and somatic cell count, expressed as linear score (LS), in milk significantly increased in Hot compared with Cold season. Coagulase-positive staphylococci on teat swabs showed higher counts in Cold season in comparison with the other ones. The effect of cow cleanliness was significant for SPC, psychrotrophic bacterial count (PBC), CC and Escherichia coli in bulk tank milk. Somatic cell count showed a relationship with udder hygiene score. Milking operation routine strongly affected bacterial counts and LS of bulk tank milk: farms that accomplished a comprehensive milking scheme including two or more operations among forestripping, pre-dipping and post-dipping had lower teat contamination and lower milk SPC, PBC, LPC, CC and LS than farms that did not carry out any operation.

  11. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    OpenAIRE

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysacc...

  12. An experimental study of Au removal from solution by non-metabolizing bacterial cells and their exudates

    Science.gov (United States)

    Kenney, Janice P. L.; Song, Zhen; Bunker, Bruce A.; Fein, Jeremy B.

    2012-06-01

    In this study, we examine the initial interactions between aqueous Au(III)-hydroxide-chloride aqueous complexes and bacteria by measuring the effects of non-metabolizing cells on the speciation and distribution of Au. We conducted batch Au(III) removal experiments, measuring the kinetics and pH dependence of Au removal, and tracking valence state transformations and binding environments using XANES spectroscopy. These experiments were conducted using non-metabolizing cells of Bacillus subtilis or Pseudomonas putida suspended in a 5 ppm Au(III)-(hydroxide)-chloride starting solution of 0.1 M NaClO4 to buffer ionic strength. Both bacterial species removed greater than 85% of the Au from solution after 2 h of exposure time below approximately pH 5. Above pH 5, the extent of Au removed from solution decreased with increasing pH, with less than approximately 10% removal of Au from solution above pH 7.5. Kinetics experiments indicated that the Au removal with both bacterial species was rapid at pH 3, and slowed with increasing pH. Reversibility experiments demonstrated that (1) once the Au was removed from solution, adjusting 35 the pH alone did not remobilize the Au into solution and (2) the presence of cysteine in solution in the reversibility experiments caused Au to desorb, suggesting that the Au was not internalized within the bacterial cells. Our results suggest that Au removal occurs as a two-step pH-dependent adsorption reduction process. The speciation of the aqueous Au and the bacterial surface appears to control the rate of Au removal from solution. Under low pH conditions, the cell walls are only weakly negatively charged and aqueous Au complexes adsorb readily and rapidly. With increasing pH, the cell wall becomes more negatively charged, slowing adsorption significantly. The XANES data demonstrate that the reduction of Au(III) by bacterial exudates is slower and less extensive than the reduction observed in the bacteria-bearing systems, and we conclude that

  13. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    Directory of Open Access Journals (Sweden)

    Mari Narusaka

    Full Text Available Housaku Monogatari (HM is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.

  14. Cell-to-Cell Propagation of the Bacterial Toxin CNF1 via Extracellular Vesicles: Potential Impact on the Therapeutic Use of the Toxin

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    Alessia Fabbri

    2015-11-01

    Full Text Available Eukaryotic cells secrete extracellular vesicles (EVs, either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1. Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

  15. Prophylaxis with levofloxacin: impact on bacterial susceptibility and epidemiology in a hematopoietic stem cell transplant unit

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    Livia Amaral Alonso Lopes

    2014-01-01

    Full Text Available Background: The emergence of resistance has been demonstrated in cancer treatment centers where prophylaxis with fluoroquinolone is used. Objective: Considering the importance of epidemiological monitoring as a strategy in choosing protocols involving antibiotics, this study aimed to evaluate the emergence of quinolone resistance and changes in the local epidemiology in a hematopoietic stem cell transplant service. Methods: For this study, 60 positive cultures before the prophylactic use of levofloxacin (period A: 2007-2008 and 118 cultures after starting the use of prophylactic levofloxacin (period B: 2010-2011 were evaluated. Results: Resistance increased for all the different types of bacteria isolated (from 46.0% to 76.5%; p-value = 0.0002. Among Gram-negative bacteria, resistance increased from 21.4% to 60.7% (p-value = 0.0163 and among Gram-positive bacteria, it increased from 55.6% to 82.9% (p-value = 0.0025. The use of levofloxacin increased from 19.44 defined daily doses per 1,000 patient-days in period A to 166.64 in period B. The use of broad spectrum antibiotics remained unchanged. Considering bacteria associated with infection, 72 and 76 were isolated in periods A and B, respectively. There was a reduction in the rate of Gramnegative bacteria in cultures associated with infection (3.81 vs. 2.00 cultures/1,000 patientdays; p-value = 0.008. Conclusion: The study of prophylaxis with levofloxacin demonstrated that there was a decrease in infections by Gram-negative bacteria; however, bacterial resistance increased, even though the use of broad-spectrum antibiotics remained unchanged. Constant monitoring of local epidemiology combined with research on clinical outcomes is needed to evaluate the effectiveness of prophylaxis.

  16. Pili Binding to Asialo-GM1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalization by Pseudomonas aeruginosa

    OpenAIRE

    Comolli, James C; Waite, Leslie L.; Keith E Mostov; Joanne N. Engel

    1999-01-01

    The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to...

  17. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation.

    Science.gov (United States)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M; Gil, Francisco J; Rodriguez, Daniel

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria-cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties.

  18. A LytM Domain Dictates the Localization of Proteins to the Mother Cell-Forespore Interface during Bacterial Endospore Formation▿ †

    OpenAIRE

    Meisner, Jeffrey; Moran, Charles P.

    2010-01-01

    A large number of proteins are known to reside at specific subcellular locations in bacterial cells. However, the molecular mechanisms by which many of these proteins are anchored at these locations remains unclear. During endospore formation in Bacillus subtilis, several integral membrane proteins are located specifically at the interface of the two adjacent cells of the developing sporangium, the mother cell and forespore. The mother cell membrane protein SpoIIIAH recognizes the cell-cell i...

  19. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

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    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  20. Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha- versus Resilon-Induced Human Monocyte Cell Line Toxicity.

    Directory of Open Access Journals (Sweden)

    Jamshid Hadjati

    2015-04-01

    Full Text Available Cytotoxic effects of obturation materials were tested in presence and absence of endotoxin on human monocytes in vitro.Human monocytes from THP-1 cell line were cultured. Three millimeters from the tip of each Resilon and gutta percha points were cut and directly placed at the bottom of the culture wells. Cultured cells were exposed to gutta percha (groups G1 and G2 and Resilon (R1 and R2. Ten μg/ml bacterial lipopolysaccharide (LPS was added to the culture wells in groups G1 and R1. Positive control included the bacterial LPS without the root canal filling material and the negative control contained the cells in culture medium only. Viability of cells was tested in all groups after 24, 48, and 72 hours using the methylthiazolyldiphenyl-tetrazolium bromide (MTT assay for at least 3 times to obtain reproducible results. Optical density values were read and the data were analyzed using three-way ANOVA and post hoc statistical test.The results showed that cells in G2 had the lowest rate of viability at 24 hours, but the lowest rate of viable cells was recorded in G1 at 48 and 72 hours. The effect of LPS treatment was not statistically significant. Resilon groups showed cell viability values higher than those of gutta percha groups, although statistically non-significant (P=0.105. Cell viability values were lower in gutta percha than Resilon groups when LPS-treated and LPS-untreated groups were compared independently at each time point.It could be concluded that none of the tested root canal filling materials had toxic effects on cultured human monocyte cells whether in presence or absence of LPS contamination.

  1. Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.

    Science.gov (United States)

    Naz, Neveda; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

    2013-01-01

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells. PMID:24192850

  2. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  3. Possible implication of bacterial infection in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Shigeo eFuji

    2014-04-01

    Full Text Available Graft-versus-host disease (GVHD is still one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT. In the pathogenesis of acute GVHD, it has been established that donor-derived T cells activated in the recipient play a major role in GVHD in initiation and maintenance within an inflammatory cascade. To reduce the risk of GVHD, intensification of GVHD prophylaxis like T cell depletion is effective, but it inevitably increases the risk of infectious diseases and abrogates beneficial graft-versus-leukemia effects. Although various cytokines are considered to play an important role in the pathogenesis of GVHD, GVHD initiation is such a complex process that cannot be prevented by means of single inflammatory cytokine inhibition. Thus, efficient methods to control the whole inflammatory milieu both on cellular and humoral view are needed. In this context, infectious diseases can theoretically contribute to an elevation of inflammatory cytokines after allogeneic HSCT and activation of various subtypes of immune effector cells, which might in summary lead to an aggravation of acute GVHD. The appropriate treatments or prophylaxis of bacterial infection during the early phase after allogeneic HSCT might be beneficial to reduce not only infectious-related but also GVHD-related mortality. Here, we aim to review the literature addressing the interactions of bacterial infections and GVHD after allogeneic HSCT.

  4. Vimentin in Bacterial Infections

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    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs. IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  5. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.

    Science.gov (United States)

    Flaherty, Rebecca A; Lee, Shaun W

    2016-08-19

    Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

  6. Nanoscale Cell Wall Deformation Impacts Long-Range Bacterial Adhesion Forces on Surfaces

    NARCIS (Netherlands)

    Chen, Yun; Harapanahalli, Akshay K.; Busscher, Henk J.; Norde, Willem; van der Mei, Henny C.

    2014-01-01

    Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of

  7. Nanoscale cell wall deformation impacts long-range bacterial adhesion forces on surfaces

    NARCIS (Netherlands)

    Chen, Y.; Harapanahalli, A.K.; Busscher, H.J.; Norde, W.; Mei, van der H.C.

    2014-01-01

    Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of

  8. Degradation of lucerne stem cell walls by five rumen bacterial species

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.; Weimer, P.J.

    2004-01-01

    The rumen bacterial strains Butyrivibrio fibrisolvens H17c, Fibrobacter succinogenes S85, Lachnospira multiparus 40, Ruminococcus albus 7 and R. flavefaciens FD-1 were compared individually and as a five-species mixture with a rumen inoculum for their ability to degrade lucerne (Medicago sativa L.)

  9. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers

    Directory of Open Access Journals (Sweden)

    Stella W. Nowotarska

    2014-06-01

    Full Text Available Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of the naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde and 2-hydroxy-5-methoxybenzaldehyde, and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be active against both Gram-positive and Gram-negative pathogenic microorganisms. The lipid monolayers consist of 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE, 1,2-dihexa- decanoyl-sn-glycero-3-phospho-(1'-rac-glycerol (DPPG, and 1,1',2,2'-tetratetradecanoyl cardiolipin (cardiolipin. Surface pressure–area (π-A and surface potential–area (Δψ-A isotherms were measured to monitor changes in the thermodynamic and physical properties of the lipid monolayers. Results of the study indicated that the five compounds modified the three lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial –lipid complexes, reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer membrane model. The interactions of the five antimicrobial compounds with bacterial phospholipids depended on both the structure of the antimicrobials and the composition of the monolayers. The observed experimental results provide insight into the mechanism of the molecular interactions between naturally-occurring antimicrobial compounds and phospholipids of the bacterial cell membrane that govern activities.

  10. Spatial distribution of bacterial communities on volumetric and planar anodes in single-chamber air-cathode microbial fuel cells

    KAUST Repository

    Vargas, Ignacio T.

    2013-05-29

    Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air cathode. As expected, the stable operational voltage and the coulombic efficiency (CE) were higher for the volumetric anode than the planar anode (0.57V and CE=22% vs. 0.51V and CE=12%). The genus Geobacter was the only known exoelectrogen among the observed dominant groups, comprising 57±4% of recovered sequences for the brush and 27±5% for the carbon-cloth anode. While the bacterial communities differed between the two anode materials, results showed that Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems. © 2013 Wiley Periodicals, Inc.

  11. Spatial distribution of bacterial communities on volumetric and planar anodes in single-chamber air-cathode microbial fuel cells.

    Science.gov (United States)

    Vargas, Ignacio T; Albert, Istvan U; Regan, John M

    2013-11-01

    Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air cathode. As expected, the stable operational voltage and the coulombic efficiency (CE) were higher for the volumetric anode than the planar anode (0.57 V and CE = 22% vs. 0.51 V and CE = 12%). The genus Geobacter was the only known exoelectrogen among the observed dominant groups, comprising 57 ± 4% of recovered sequences for the brush and 27 ± 5% for the carbon-cloth anode. While the bacterial communities differed between the two anode materials, results showed that Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems. PMID:23616357

  12. A channel connecting the mother cell and forespore during bacterial endospore formation

    OpenAIRE

    Meisner, Jeffrey; Xin WANG; Serrano, Monica; Henriques, Adriano O.; Moran, Charles P.

    2008-01-01

    At an early stage during Bacillus subtilis endospore development the bacterium divides asymmetrically to produce two daughter cells. The smaller cell (forespore) differentiates into the endospore, while the larger cell (mother cell) becomes a terminally differentiated cell that nurtures the developing forespore. During development the mother cell engulfs the forespore to produce a protoplast, surrounded by two bilayer membranes, which separate it from the cytoplasm of the mother cell. The act...

  13. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    Science.gov (United States)

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  14. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    Science.gov (United States)

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

  15. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.;

    ) coatings on titanium. We investigate the ability of a high density poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coating to resist bacterial adhesion and biofilm formation from three clinically relevant bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermis. The high...... density PLL-g-PEG coatings were about eight times as thick as the conventional PLL-g-PEG coatings. Adhesion forces toward high density PLL-g-PEG coatings were low (P. aeruginosa) or close to zero (S. aureus and S. epidermidis) compared to bare titanium surface. However, no decrease in adhesion force...... was observed for S. epidermidis toward conventional PLL-g-PEG coatings, whereas significantly lower adhesion forces were observed for S. aureus and P. aeruginosa. The adhesion force patterns were reflected by the colonization of bacteria after 48 h incubation of the coatings in bacterial cultures. The high...

  16. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    OpenAIRE

    Albrecht A.; Rafrafi Y.; Sablayrolles C.; Bertron A.; Kassim C.; Alquier M.; Erable B.

    2013-01-01

    International audience Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 < pH < 11). The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bac...

  17. Procalcitonin Identifies Cell Injury, Not Bacterial Infection, in Acute Liver Failure.

    Directory of Open Access Journals (Sweden)

    Jody A Rule

    Full Text Available Because acute liver failure (ALF patients share many clinical features with severe sepsis and septic shock, identifying bacterial infection clinically in ALF patients is challenging. Procalcitonin (PCT has proven to be a useful marker in detecting bacterial infection. We sought to determine whether PCT discriminated between presence and absence of infection in patients with ALF.Retrospective analysis of data and samples of 115 ALF patients from the United States Acute Liver Failure Study Group randomly selected from 1863 patients were classified for disease severity and ALF etiology. Twenty uninfected chronic liver disease (CLD subjects served as controls.Procalcitonin concentrations in most samples were elevated, with median values for all ALF groups near or above a 2.0 ng/mL cut-off that generally indicates severe sepsis. While PCT concentrations increased somewhat with apparent liver injury severity, there were no differences in PCT levels between the pre-defined severity groups-non-SIRS and SIRS groups with no documented infections and Severe Sepsis and Septic Shock groups with documented infections, (p = 0.169. PCT values from CLD patients differed from all ALF groups (median CLD PCT value 0.104 ng/mL, (p ≤0.001. Subjects with acetaminophen (APAP toxicity, many without evidence of infection, demonstrated median PCT >2.0 ng/mL, regardless of SIRS features, while some culture positive subjects had PCT values <2.0 ng/mL.While PCT appears to be a robust assay for detecting bacterial infection in the general population, there was poor discrimination between ALF patients with or without bacterial infection presumably because of the massive inflammation observed. Severe hepatocyte necrosis with inflammation results in elevated PCT levels, rendering this biomarker unreliable in the ALF setting.

  18. Commensal bacterial internalization by epithelial cells: An alternative portal for gut leakiness

    OpenAIRE

    Yu, Linda Chia-Hui

    2015-01-01

    Co-existing paracellular and transcellular barrier defect in intestinal epithelium was documented in inflammatory bowel disease, celiac disease, and intestinal obstruction. Mechanisms regarding tight junction disruption have been extensively studied; however, limited progress has been made in research on bacterial transcytosis. Densely packed brush border (BB), with cholesterol-based lipid rafts in the intermicrovillous membrane invagination, serves as an ultrastructural barrier to prevent di...

  19. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Science.gov (United States)

    Alekseeva, Ludmila; Rault, Lucie; Almeida, Sintia; Legembre, Patrick; Edmond, Valérie; Azevedo, Vasco; Miyoshi, Anderson; Even, Sergine; Taieb, Frédéric; Arlot-Bonnemains, Yannick; Le Loir, Yves; Berkova, Nadia

    2013-01-01

    Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  20. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Directory of Open Access Journals (Sweden)

    Ludmila Alekseeva

    Full Text Available Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  1. Molecular mechanism of immune cells activated by bacterial DNA%细菌DNA激活免疫细胞的分子机制

    Institute of Scientific and Technical Information of China (English)

    王良喜; 周红

    2003-01-01

    Bacterial DNA taken up by immune cells in a CpG motif- independent manner is translo-cated into endosome. Endosomal maturation is essential for subsequent bacterial DNA - mediated signal trans-duction. TLR9 is recruited into endosome to recognize bacterial DNA and initiate the TLB/IL- 1R signal transduction pathway. As a result , transcription factors NF - κB and AP- 1 are activated, which, in tum,leads to proinflammatory cytokine expression and induces a strong acute Th1 - like inflammatory response.

  2. Respiratory Viruses Augment the Adhesion of Bacterial Pathogens to Respiratory Epithelium in a Viral Species- and Cell Type-Dependent Manner

    OpenAIRE

    Avadhanula, Vasanthi; Rodriguez, Carina A.; DeVincenzo, John P.; Wang, Yan; Webby, Richard J; Ulett, Glen C.; Adderson, Elisabeth E.

    2006-01-01

    Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known recept...

  3. Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.

    Directory of Open Access Journals (Sweden)

    Manuel Hitzler

    Full Text Available Langerhans cells (LCs are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs, LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN or lipopolysaccharide (LPS and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4(+T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+/CCR4(+ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6(+ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6(+/CCR4(+ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.

  4. Subvisible (2-100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

    Science.gov (United States)

    Corvari, Vincent; Narhi, Linda O; Spitznagel, Thomas M; Afonina, Nataliya; Cao, Shawn; Cash, Patricia; Cecchini, Irene; DeFelippis, Michael R; Garidel, Patrick; Herre, Andrea; Koulov, Atanas V; Lubiniecki, Tony; Mahler, Hanns-Christian; Mangiagalli, Paolo; Nesta, Douglas; Perez-Ramirez, Bernardo; Polozova, Alla; Rossi, Mara; Schmidt, Roland; Simler, Robert; Singh, Satish; Weiskopf, Andrew; Wuchner, Klaus

    2015-11-01

    Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes. PMID:26324466

  5. Effects of elaidic acid, a predominant industrial trans fatty acid, on bacterial growth and cell surface hydrophobicity of lactobacilli.

    Science.gov (United States)

    Wu, Qinglong; Shah, Nagendra P

    2014-12-01

    The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log10 CFU/mL was still reached for tested lactobacilli after 24-h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24-h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.

  6. Effects of inoculation sources on the enrichment and performance of anode bacterial consortia in sensor typed microbial fuel cells

    Directory of Open Access Journals (Sweden)

    Phuong Tran

    2016-01-01

    Full Text Available Microbial fuel cells are a recently emerging technology that promises a number of applications in energy recovery, environmental treatment and monitoring. In this study, we investigated the effect of inoculating sources on the enrichment of electrochemically active bacterial consortia in sensor-typed microbial fuel cells (MFCs. Several MFCs were constructed, operated with modified artificial wastewater and inoculated with different microbial sources from natural soil, natural mud, activated sludge, wastewater and a mixture of those sources. After enrichment, the MFCs inoculated with the natural soil source generated higher and more stable currents (0.53±0.03 mA, in comparisons with the MFCs inoculated with the other sources. The results from denaturing gradient gel electrophoresis (DGGE showed that there were significant changes in bacterial composition from the original inocula to the enriched consortia. Even more interestingly, Pseudomonas sp. was found dominant in the natural soil source and also in the corresponding enriched consortium. The interactions between Pseudomonas sp. and other species in such a community are probably the key for the effective and stable performance of the MFCs.

  7. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  8. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  9. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

    DEFF Research Database (Denmark)

    Rosenstiel, Philip; Sina, Christian; End, Caroline;

    2007-01-01

    -kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology......Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes...... intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF...

  10. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

    OpenAIRE

    Mistou, Michel-Yves; Sutcliffe, Iain; van Sorge, Nina

    2016-01-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall te...

  11. Addition of selenium nanoparticles to electrospun silk scaffolds improves mammalian cell activity while reducing bacterial growth

    Directory of Open Access Journals (Sweden)

    Stanley Chung

    2016-07-01

    Full Text Available Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas compared to non-electrospun equivalents. However, purified silk promotes microbial growth. In contrast, selenium nanoparticles have excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics.

  12. Bacterial Hydrodynamics

    Science.gov (United States)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  13. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  14. Temporal trends in bulk tank somatic cell count and total bacterial count in Irish dairy herds during the past decade.

    Science.gov (United States)

    Berry, D P; O'Brien, B; O'Callaghan, E J; Sullivan, K O; Meaney, W J

    2006-10-01

    The objective of this study was to document temporal trends in bulk tank somatic cell count (SCC) and total bacterial counts (TBC) in Irish dairy herds during the years 1994 to 2004. Three milk processors participated in the study, providing data on 2,754,270 individual bulk tank SCC and 2,056,992 individual bulk tank TBC records from 9,113 herds. Somatic cell counts decreased during the years 1994 to 2000, followed by an annual increase thereafter of more than 2,000 cells/mL. A tendency existed for TBC to decrease over time. Across all years, bulk tank SCC were the lowest in April and highest in November; TBC were the lowest in May and highest in December. The significant seasonal pattern observed in herd SCC and TBC was an artifact of seasonal calving in Ireland. In general, herds selling more milk had lower bulk tank SCC and TBC. Herds having the highest SCC (i.e., > 450,000 cells/mL) and the lowest SCC (i.e., < or = 150,000 cells/mL) both contributed substantially to the mean SCC of the milk pool collected by the milk processors. Derived transition matrices showed that between adjacent years, herds had the greatest probability of remaining in the same annual mean SCC or TBC category.

  15. Metal ion effects on hydraulic conductivity of bacterial cellulose-pectin composites used as plant cell wall analogs.

    Science.gov (United States)

    McKenna, Brigid A; Kopittke, Peter M; Wehr, J Bernhard; Blamey, F Pax C; Menzies, Neal W

    2010-02-01

    Low concentrations of some trace metals markedly reduce root elongation rate and cause ruptures to root rhizodermal and outer cortical cells in the elongation zone. The interactions between the trace metals and plant components responsible for these effects are not well understood but may be linked to changes in water uptake, cell turgor and cell wall extensibility. An experiment was conducted to investigate the effects of Al, La, Cu, Gd, Sc and Ru on the saturated hydraulic conductivity of bacterial cellulose (BC)-pectin composites, used as plant cell wall analogs. Hydraulic conductivity was reduced to approximately 30% of the initial flow rate by 39 microM Al and 0.6 microM Cu, approximately 40% by 4.6 microM La, 3 microM Sc and 4.4 microM Ru and approximately 55% by 3.4 microM Gd. Scanning electron microscopy (SEM) revealed changes in the ultrastructure of the composites. The results suggest that trace metal binding decreases the hydraulic conductivity through changes in pectin porosity. The experiment illustrates the importance of metal interactions with pectin, and the implications of such an interaction in plant metal toxicity and in normal cell wall processes. PMID:20053181

  16. Bacterial Wall Components such as Lipothecoid Acid, Peptidoglycan, Liposaccharide and Lipid A Stimulate Cell Proliferation in Intestinal Epithelial Cells

    OpenAIRE

    Olaya, Jaime H.; Neopikhanov, Vadim; Söderman, Charlotte; Uribe, Andrés

    2011-01-01

    Earlier studies indicate that the microflora contains mitogens to intestinal epithelial cells. Our aim is to examine whether cell wall components of both Gram-negative and positive bacteria influence cell proliferation in small intestinal and colonic epithelial cells. A human colonic epithelial cell line from adenocarcinoma (IEC-6) and a nontransformed small intestinal cell line from germ-free rats (LS-123) were incubated with (a) lipothecoid acid from Streptococcus faecalis at 1.56–50 ...

  17. Moxifloxacin in Preventing Bacterial Infections in Patients Who Have Undergone Donor Stem Cell Transplant

    Science.gov (United States)

    2013-03-14

    Breast Cancer; Chronic Myeloproliferative Disorders; Gestational Trophoblastic Tumor; Infection; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasms; Neuroblastoma; Ovarian Cancer; Testicular Germ Cell Tumor

  18. Mucin biopolymers prevent bacterial aggregation by retaining cells in the free-swimming state

    Science.gov (United States)

    Caldara, Marina; Friedlander, Ronn S.; Kavanaugh, Nicole L.; Aizenberg, Joanna; Foster, Kevin R.; Ribbeck, Katharina

    2013-01-01

    Summary Many species of bacteria form surface-attached communities known as biofilms. Surrounded in secreted polymers, these aggregates are difficult to both prevent and eradicate, posing problems for medicine and industry [1, 2]. Humans play host to hundreds of trillions of microbes that live adjacent to our epithelia and we are typically able to prevent harmful colonization. Mucus, the hydrogel overlying all wet epithelia in the body, can prevent bacterial contact with the underlying tissue. The digestive tract, for example, is lined by a firmly adherent mucus layer that is typically devoid of bacteria, followed by a second, loosely adherent layer that contains numerous bacteria [3]. Here, we investigate mucus's role as a principle arena for host-microbe interactions. Using defined in vitro assays, we found that mucin biopolymers, the main functional constituents of mucus, promote the motility of planktonic bacteria, and prevent their adhesion to underlying surfaces. The deletion of motility genes, however, allows Pseudomonas aeruginosa to overcome the dispersive effects of mucus and form suspended antibiotic-resistant flocs, which mirror the clustered morphology of immotile natural isolates found in the cystic fibrosis lung mucus [4, 5]. Mucus may offer new strategies to target bacterial virulence, such as the design of anti-biofilm coatings for implants. PMID:23142047

  19. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    International Nuclear Information System (INIS)

    Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 < pH <11). The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours) and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions. (authors)

  20. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Directory of Open Access Journals (Sweden)

    Albrecht A.

    2013-07-01

    Full Text Available Leaching experiments of solid matrices (bitumen and cement pastes have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.. Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  1. Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces

    Science.gov (United States)

    Monier, J.-M.; Lindow, S. E.

    2003-01-01

    The survival of individual Pseudomonas syringae cells was determined on bean leaf surfaces maintained under humid conditions or periodically exposed to desiccation stress. Cells of P. syringae strain B728a harboring a GFP marker gene were visualized by epifluorescence microscopy, either directly in situ or after recovery from leaves, and dead cells were identified as those that were stained with propidium iodide in such populations. Under moist, conducive conditions on plants, the proportion of total live cells was always high, irrespective of their aggregated state. In contrast, the proportion of the total cells that remained alive on leaves that were periodically exposed to desiccation stress decreased through time and was only ≈15% after 5 days. However, the fraction of cells in large aggregates that were alive on such plants in both condition was much higher than more solitary cells. Immediately after inoculation, cells were randomly distributed over the leaf surface and no aggregates were observed. However, a very aggregated pattern of colonization was apparent within 7 days, and >90% of the living cells were located in aggregates of 100 cells or more. Our results strongly suggest that, although conducive conditions favor aggregate formation, such cells are much more capable of tolerating environmental stresses, and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces. PMID:14665692

  2. An X-ray Absorption Fine Structure study of Au adsorbed onto the non-metabolizing cells of two soil bacterial species

    Science.gov (United States)

    Song, Zhen; Kenney, Janice P. L.; Fein, Jeremy B.; Bunker, Bruce A.

    2012-06-01

    Gram-positive and Gram-negative bacterial cells can remove Au from Au(III)-chloride solutions, and the extent of removal is strongly pH dependent. In order to determine the removal mechanisms, X-ray Absorption Fine Structure (XAFS) spectroscopy experiments were conducted on non-metabolizing biomass of Bacillus subtilis and Pseudomonas putida with fixed Au(III) concentrations over a range of bacterial concentrations and pH values. X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data on both bacterial species indicate that more than 90% of the Au atoms on the bacterial cell walls were reduced to Au(I). In contrast to what has been observed for Au(III) interaction with metabolizing bacterial cells, no Au(0) or Au-Au nearest neighbors were observed in our experimental systems. All of the removed Au was present as adsorbed bacterial surface complexes. For both species, the XAFS data suggest that although Au-chloride-hydroxide aqueous complexes dominate the speciation of Au in solution, Au on the bacterial cell wall is characterized predominantly by binding of Au atoms to sulfhydryl functional groups and amine and/or carboxyl functional groups, and the relative importance of the sulfhydryl groups increases with increasing pH and with decreasing Au loading. The XAFS data for both microorganism species suggest that adsorption is the first step in the formation of Au nanoparticles by bacteria, and the results enhance our ability to account for the behavior of Au in bacteria-bearing geologic systems.

  3. An X-ray Absorption Fine Structure study of Au adsorbed onto the non-metabolizing cells of two soil bacterial species

    Energy Technology Data Exchange (ETDEWEB)

    Song, Zhen; Kenney, Janice P.L.; Fein, Jeremy B.; Bunker, Bruce A. (Notre)

    2015-02-09

    Gram-positive and Gram-negative bacterial cells can remove Au from Au(III)-chloride solutions, and the extent of removal is strongly pH dependent. In order to determine the removal mechanisms, X-ray Absorption Fine Structure (XAFS) spectroscopy experiments were conducted on non-metabolizing biomass of Bacillus subtilis and Pseudomonas putida with fixed Au(III) concentrations over a range of bacterial concentrations and pH values. X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data on both bacterial species indicate that more than 90% of the Au atoms on the bacterial cell walls were reduced to Au(I). In contrast to what has been observed for Au(III) interaction with metabolizing bacterial cells, no Au(0) or Au-Au nearest neighbors were observed in our experimental systems. All of the removed Au was present as adsorbed bacterial surface complexes. For both species, the XAFS data suggest that although Au-chloride-hydroxide aqueous complexes dominate the speciation of Au in solution, Au on the bacterial cell wall is characterized predominantly by binding of Au atoms to sulfhydryl functional groups and amine and/or carboxyl functional groups, and the relative importance of the sulfhydryl groups increases with increasing pH and with decreasing Au loading. The XAFS data for both microorganism species suggest that adsorption is the first step in the formation of Au nanoparticles by bacteria, and the results enhance our ability to account for the behavior of Au in bacteria-bearing geologic systems.

  4. Timescales and Frequencies of Reversible and Irreversible Adhesion Events of Single Bacterial Cells.

    Science.gov (United States)

    Hoffman, Michelle D; Zucker, Lauren I; Brown, Pamela J B; Kysela, David T; Brun, Yves V; Jacobson, Stephen C

    2015-12-15

    In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to

  5. Short communication: bulk tank total bacterial count in dairy sheep: factors of variation and relationship with somatic cell count.

    Science.gov (United States)

    Gonzalo, C; Carriedo, J A; Beneitez, E; Juárez, M T; De La Fuente, L F; San Primitivo, F

    2006-02-01

    A total of 9,353 records for bulk tank total bacterial count (TBC) were obtained over 1 yr from 315 dairy ewe flocks belonging to the Sheep Improvement Consortium (CPO) in Castilla-León (Spain). Analysis of variance showed significant effects of flock, breed, month within flock, dry therapy, milking type and installation, and logSCC on logTBC. Flock and month within flock were important variation factors as they accounted for 22.0 and 22.1% of the variance, respectively. Considerable repeatability values were obtained for both random factors. Hand milking and bucket-milking machines elicited highest logTBC (5.31), whereas parlor systems with looped milkline (5.01) elicited the lowest logTBC. The implementation of dry therapy practice (5.12) showed significantly lower logTBC than when not used (5.25). Variability in logTBC among breeds ranged from 5.24 (Awassi) to 5.07 (Churra). However, clinical outbreaks of contagious agalactia did not increase TBC significantly. A statistically significant relationship was found between logTBC and logSCC, the correlation coefficient between the variables being r = 0.23. Programs for improving milk hygiene should be implemented for both total bacterial count and somatic cell count variables at the same time.

  6. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Matera Giovanni

    2012-05-01

    Full Text Available Abstract Background Procalcitonin (PCT is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS, reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p Salmonella typhimurium (rough chemotype and Escherichia coli (smooth chemotype. Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC. When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10 and tumor necrosis factor alpha (TNFα by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1 exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators.

  7. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Yam, Joey Kuok Hoong;

    2014-01-01

    Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from...... biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression...... of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a...

  8. Microfluidic-Based Droplet and Cell Manipulations Using Artificial Bacterial Flagella

    Directory of Open Access Journals (Sweden)

    Yun Ding

    2016-02-01

    Full Text Available Herein, we assess the functionality of magnetic helical microswimmers as basic tools for the manipulation of soft materials, including microdroplets and single cells. Their ability to perform a range of unit operations is evaluated and the operational challenges associated with their use are established. In addition, we also report on interactions observed between the head of such helical swimmers and the boundaries of droplets and cells and discuss the possibilities of assembling an artificial swimming microorganism or a motorized cell.

  9. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

    Science.gov (United States)

    Wong, Connie H Y; Jenne, Craig N; Petri, Björn; Chrobok, Navina L; Kubes, Paul

    2013-08-01

    Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

  10. Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

    Science.gov (United States)

    Katz, Assaf; Elgamal, Sara; Rajkovic, Andrei; Ibba, Michael

    2016-08-01

    Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.

  11. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

    Directory of Open Access Journals (Sweden)

    Wilhelm T. S. Huck

    2013-05-01

    Full Text Available In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl-L-homoserine lactone (OdDHL. Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

  12. Bacterial cellulose-polyaniline nano-biocomposite: A porous media hydrogel bioanode enhancing the performance of microbial fuel cell

    Science.gov (United States)

    Mashkour, Mehrdad; Rahimnejad, Mostafa; Mashkour, Mahdi

    2016-09-01

    Microbial fuel cells (MFCs) are one of the possible renewable energy supplies which microorganisms play an active role in bio-oxidize reactions of a substrate such as glucose. Electrode materials and surface modifications are highly effective tools in enhancing MFCs' Performance. In this study, new composite anodes are fabricated. Bacterial cellulose (BC) is used as continuous phase and polyaniline (PANI) as dispersed one which is synthesized by in situ chemical oxidative polymerization on BC's fibers. With hydrogel nature of BC as a novel feature and polyaniline conductivity there meet the favorable conditions to obtain an active microbial biofilm on anode surface. Maximum power density of 117.76 mW/m2 in current density of 617 mA/m2 is achieved for BC/PANI anode. The amounts demonstrate a considerable enhancement compared with graphite plate (1 mW/m2 and 10 mA/m2).

  13. Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts

    Science.gov (United States)

    Luan, Jun-Bo; Shan, Hong-Wei; Isermann, Philipp; Huang, Jia-Hsin; Lammerding, Jan; Liu, Shu-Sheng; Douglas, Angela E.

    2016-01-01

    Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell–cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell–cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends. PMID:27358364

  14. B and T cell crosstalk in anti-bacterial immune responses

    NARCIS (Netherlands)

    J. de Wit

    2012-01-01

    This thesis shows that phagocytosis of Salmonella by B cells may generate a survival niche and transport vehicle for Salmonella, but that simultaneously Salmonella-infected B cells induce an optimal anti-Salmonella response through activation of multiple arms of the adaptive immune response. The the

  15. Mutual regulation causes co-entrainment between a synthetic oscillator and the bacterial cell cycle.

    Science.gov (United States)

    Dies, Marta; Galera-Laporta, Leticia; Garcia-Ojalvo, Jordi

    2016-04-18

    The correct functioning of cells requires the orchestration of multiple cellular processes, many of which are inherently dynamical. The conditions under which these dynamical processes entrain each other remain unclear. Here we use synthetic biology to address this question in the case of concurrent cellular oscillations. Specifically, we study at the single-cell level the interaction between the cell division cycle and a robust synthetic gene oscillator in Escherichia coli. Our results suggest that cell division is able to partially entrain the synthetic oscillations under normal growth conditions, by driving the periodic replication of the genes involved in the oscillator. Coupling the synthetic oscillations back into the cell cycle via the expression of a key regulator of chromosome replication increases the synchronization between the two periodic processes. A simple computational model allows us to confirm this effect.

  16. Arthritis by autoreactive T cell lines obtained from rats after injection of intestinal bacterial cell wall fragments

    NARCIS (Netherlands)

    I. Klasen (Ina); J. Kool (Jeanette); M.J. Melief; I. Loeve (I.); W.B. van den Berg (Wim); A.J. Severijnen; M.P.H. Hazenberg (Maarten)

    1992-01-01

    markdownabstract__Abstract__ T cell lines (B13, B19) were isolated from the lymph nodes of Lewis rats 12 days after an arthritogenic injection of cell wall fragments of Eubacterium aerofaciens (ECW), a major resident of the human intestinal flora. These cell wall fragments consist of peptidoglycan

  17. Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

    Science.gov (United States)

    Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

  18. Efficacy of coating activated carbon with milk proteins to prevent binding of bacterial cells from foods for PCR detection.

    Science.gov (United States)

    Opet, Nathan J; Levin, Robert E

    2013-08-01

    Foods contaminated with pathogens are common sources of illness. Currently, the most common and sensitive rapid detection method involves the PCR. However, food matrices are complex and contain inhibitors that limit the sensitivity of the PCR. The use of coated activated carbon can effectively facilitate the removal of PCR inhibitors without binding targeted bacterial cells from food samples. With the use of activated carbon coated with milk proteins, a cell recovery at pH 7.0 of 95.7%±2.0% was obtained, compared to control uncoated activated carbon, which yielded a cell recovery of only 1.1%±0.8%. In addition, the milk protein coated activated carbon was able to absorb similar amounts of soluble compounds as uncoated activated carbon, with the exception of bovine hemoglobin. This suggests that the use of milk proteins to coat activated carbon may therefore serve as a suitable replacement for bentonite in the coating of activated carbon, which has previously been used for the removal of PCR inhibitors from food.

  19. The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

    Science.gov (United States)

    Sundararajan, Kousik; Miguel, Amanda; Desmarais, Samantha M; Meier, Elizabeth L; Casey Huang, Kerwyn; Goley, Erin D

    2015-01-01

    The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment. PMID:26099469

  20. CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

    Science.gov (United States)

    Shen, Haiqian; Gonzalez-Juarbe, Norberto; Blanchette, Krystle; Crimmins, Gregory; Bergman, Molly A; Isberg, Ralph R; Orihuela, Carlos J; Dube, Peter H

    2016-09-01

    CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death. PMID:27268148

  1. CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

    Science.gov (United States)

    Shen, Haiqian; Gonzalez-Juarbe, Norberto; Blanchette, Krystle; Crimmins, Gregory; Bergman, Molly A; Isberg, Ralph R; Orihuela, Carlos J; Dube, Peter H

    2016-09-01

    CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.

  2. Effect of dietary bovine colostrum on the responses of immune cells to stimulation with bacterial lipopolysaccharide.

    Science.gov (United States)

    Xu, Mei Ling; Kim, Hyoung Jin; Kim, Hong-Jin

    2014-04-01

    Previous studies have revealed that ingestion of bovine colostrum is effective in preventing pathogens from invading through the gastrointestinal tract (GI) and modulating the mucosal immunity of the GI tract, indicating that its effect is principally local. Thus it is unclear if ingestion of bovine colostrum can affect the systemic immune system. In this study, we investigated the effect of taking bovine colostrum (vs phosphate-buffered saline) for 14 days on the behavior of the immune cells of mice. Isolated splenocytes, which are pivotal cells of systemic immunity, were then stimulated with Escherichia coli lipopolysaccharide. Bovine colostrum significantly reduced NK cell and monocyte activities and lymphoproliferaltive responses to LPS stimulation. Thus dietary bovine colostrum renders immune cells less responsive to LPS stimulation. Dietary bovine colostrum thus affects the systemic immune system and may have anti-inflammatory actions. PMID:24234910

  3. Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

    Science.gov (United States)

    Schaefer, M.; Zimmermann, H.; Schmitz, C.

    1994-01-01

    Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

  4. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Science.gov (United States)

    Alquier, M.; Kassim, C.; Bertron, A.; Rafrafi, Y.; Sablayrolles, C.; Albrecht, A.; Erable, B.

    2013-07-01

    Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  5. Sensitivity of bacterial biofilms and planktonic cells to a new antimicrobial agent, Oxsil 320N.

    Science.gov (United States)

    Surdeau, N; Laurent-Maquin, D; Bouthors, S; Gellé, M P

    2006-04-01

    The effective concentrations of disinfectants were determined for planktonic bacteria using the norms EN 1040 and NF T 72-150. This concentration corresponds to biocide efficacy after 5 min of contact, followed by neutralization. However, micro-organisms often colonize a substratum and form microcolonies or biofilms where they are enclosed in exopolymer matrices. Biofilms are commonly resistant to a broad range of antimicrobial agents, and resistance mechanisms involve exopolymer matrices, changes in gene expression and metabolic alterations. Due to these different resistance mechanisms, it is difficult to select and titrate antimicrobial agents to be effective against biofilms. In this context, SODIFRA developed a new disinfectant, Oxsil 320N (French patent 94 15 193). Oxsil 320N is an association of three active principles: hydrogen peroxide, acetic acid/peracetic acid and silver. This biocide was tested on planktonic bacteria and on 24-h biofilms formed on AISI 304 stainless steel surfaces. The effective concentration of Oxsil 320N was also determined on biofilms using SODIFRA recommendations (without neutralization of the biocide). Data showed that the antimicrobial efficacy measured on planktonic bacteria is not a reliable indicator of performance when biofilm is present. When biofilms were exposed to Oxsil 320N, the concentration needed to achieve a 10(5)-fold decrease in concentration was 10 times higher than that for bacterial suspensions (0.313% Oxsil 320N). An effective concentration of Oxsil 320N of 3.13% was required. PMID:16478644

  6. BIOSORPTION OF TEXTILE DYE USING IMMOBILIZED BACTERIAL (PSEUDOMONAS AERUGINOSA AND FUNGAL (PHANEROCHATE CHRYSOSPORIUM CELLS

    Directory of Open Access Journals (Sweden)

    Natarajan Saravanan

    2013-01-01

    Full Text Available Wastewater containing dyes presents a serious problem due to its high toxicity which leads to creating enormous environmental pollution and ecological hazards. Therefore the removal of the high stable dyes from the textile effluents is of major importance. The purpose of this study is to remove the reactive dye Procion Blue 2G from textile dye solution by biosorption process using immobilized cells of Pseudomonas aeruginosa and Phanerochate chrysosporium. It was found that maximum dye uptake is 1.648 mg g-1 of bead for P. aeruginosa and it is 1.242 mg g-1 of bead for P. chrysosporium. Both the results are derived from higher initial dye concentration (100 mg L-1 and high cell concentration (in terms of volume of inoculum 20 mL and at low mass of biosorbent (5 g of bead. Comparatively better results are produced by the beads having the cells of P. aeruginosa than P. chrysosporium. Further, due to the cell immobilization, both the cell beads can be utilized repeatedly in continuous reactors by selecting suitable eluent in industrial scale with the advantage of avoiding wash out of cells.

  7. Whole genome amplification and de novo assembly of single bacterial cells.

    Directory of Open Access Journals (Sweden)

    Sébastien Rodrigue

    Full Text Available BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA, and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

  8. SIMSISH technique does not alter the apparent isotopic composition of bacterial cells.

    Directory of Open Access Journals (Sweden)

    Olivier Chapleur

    Full Text Available In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH and nanoscale secondary ion mass spectrometry (nanoSIMS imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine - iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific (13C uptake during labelled methanol anaerobic degradation.

  9. In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

    Science.gov (United States)

    Smith-Palmer, Truis; Lin, Sicheng; Oguejiofor, Ikenna; Leng, Tianyang; Pustam, Amanda; Yang, Jin; Graham, Lori L; Wyeth, Russell C; Bishop, Cory D; DeMont, M Edwin; Pink, David

    2016-02-01

    Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

  10. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    Directory of Open Access Journals (Sweden)

    Bang Dang D

    2008-06-01

    Full Text Available Abstract Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C. jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process.

  11. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  12. Gene dosage imbalance during DNA replication controls bacterial cell-fate decision

    Science.gov (United States)

    Igoshin, Oleg

    Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin, the other close to the terminus leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A~P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell-cycle spent in starvation. Furthermore, changes in DNA replication and cell-cycle parameters with decreased growth rate in starvation conditions enable cells to indirectly detect starvation without the need for evaluating specific metabolites. The simplicity of the uncovered coordination mechanism and starvation sensing suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. This work is supported by National Science Foundation Grants MCB-1244135, EAGER-1450867, MCB-1244423, NIH NIGMS Grant R01 GM088428 and HHMI International Student Fellowship.

  13. Metabolic potentiation of the radiosensitization of hypoxic bacterial cells afforded by nitroaromatic compounds

    International Nuclear Information System (INIS)

    Prolonged preirradiation incubation of nitroaromatic radiosensitizers with Escherichia coli cells has been found to increase the degree of radiosensitization of the cells in anoxia. Studies with E. coli strains which differ in their nitroreductase activity indicate that the increase in sensitization arises from the action of metabolites produced by the nitroreductase system of the cell. The metabolites alone appear to decrease the extrapolation number of irradiated hypoxic cells and when combined with the parent compound give a biphasic survival curve. The combination of misonidazole (1 mmole dm-3) and its metabolites (1 mmole dm-3) gave initial and final enhancement ratios of 2.4 and 1.4, respectively. The final enhancement ratio is that expected for 1 mmole dm-3 misonidazole alone, whereas the initial enhancement ratio indicates that the metabolites potentiate the action of misonidaxole. The preirradiation incubation effect is removed by dithiothreitol at concentrations which do not affect the radiosensitization level of the nitroaromatic sensitizer. This result indicates that the active metabolite probably depletes a certain amount of the free-thiol compounds inside the cell which assist in the repair of radiation-induced damage

  14. Measurement of the copy number of the master quorum-sensing regulator of a bacterial cell.

    Science.gov (United States)

    Teng, Shu-Wen; Wang, Yufang; Tu, Kimberly C; Long, Tao; Mehta, Pankaj; Wingreen, Ned S; Bassler, Bonnie L; Ong, N P

    2010-05-19

    Quorum-sensing is the mechanism by which bacteria communicate and synchronize group behaviors. Quantitative information on parameters such as the copy number of particular quorum-sensing proteins should contribute strongly to understanding how the quorum-sensing network functions. Here, we show that the copy number of the master regulator protein LuxR in Vibrio harveyi can be determined in vivo by exploiting small-number fluctuations of the protein distribution when cells undergo division. When a cell divides, both its volume and LuxR protein copy number, N, are partitioned with slight asymmetries. We measured the distribution functions describing the partitioning of the protein fluorescence and the cell volume. The fluorescence distribution is found to narrow systematically as the LuxR population increases, whereas the volume partitioning is unchanged. Analyzing these changes statistically, we determined that N = 80-135 dimers at low cell density and 575 dimers at high cell density. In addition, we measured the static distribution of LuxR over a large (3000) clonal population. Combining the static and time-lapse experiments, we determine the magnitude of the Fano factor of the distribution. This technique has broad applicability as a general in vivo technique for measuring protein copy number and burst size. PMID:20441767

  15. Structural insights into inhibition of lipid I production in bacterial cell wall synthesis.

    Science.gov (United States)

    Chung, Ben C; Mashalidis, Ellene H; Tanino, Tetsuya; Kim, Mijung; Matsuda, Akira; Hong, Jiyong; Ichikawa, Satoshi; Lee, Seok-Yong

    2016-05-26

    Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraYAA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraYAA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraYAA. Surprisingly, MD2 does not interact with three acidic residues or the Mg(2+) cofactor required for catalysis, suggesting that MD2 binds to MraYAA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraYAA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO. PMID:27088606

  16. Scaling laws governing stochastic growth and division of single bacterial cells

    CERN Document Server

    Iyer-Biswas, Srividya; Henry, Jonathan T; Lo, Klevin; Burov, Stanislav; Lin, Yihan; Crooks, Gavin E; Crosson, Sean; Dinner, Aaron R; Scherer, Norbert F

    2014-01-01

    Uncovering the quantitative laws that govern the growth and division of single cells remains a major challenge. Using a unique combination of technologies that yields unprecedented statistical precision, we find that the sizes of individual Caulobacter crescentus cells increase exponentially in time. We also establish that they divide upon reaching a critical multiple ($\\approx$1.8) of their initial sizes, rather than an absolute size. We show that when the temperature is varied, the growth and division timescales scale proportionally with each other over the physiological temperature range. Strikingly, the cell-size and division-time distributions can both be rescaled by their mean values such that the condition-specific distributions collapse to universal curves. We account for these observations with a minimal stochastic model that is based on an autocatalytic cycle. It predicts the scalings, as well as specific functional forms for the universal curves. Our experimental and theoretical analysis reveals a ...

  17. THE TRANSMEMBRANE SIGNAL TRANSDUCTION IN HEp-2 CELLS INDUCED BY BACTERIAL ADHERENCE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ In order to understand the role of transmembrane signal transduction of host cells in the early steps of infection,the adherence of E. coli to HEp-2 cells and the change of activity of phospholipase C-γ (PLC-γ) induced by the adherence were investigated.The adherence of enteropathogenic E.coli (EPEC), strain E.7, induced a significant increase of inositol-triphosphat (IP-3) level in HEp-2 cells. The adherence of the bacteria and the increase of IP-3 was kinetically correlated. Whereas the increase of IP3 level induced by the adherence of the control strain EPEC (H511), a non-piliated strain, was much meager than that by E7, a piliated strain. The results highlighted an important role of transmembrane signals like IP-3 in the pathogenesis of EPEC.

  18. A polymerization–depolymerization model for generation of contractile force during bacterial cell division

    Indian Academy of Sciences (India)

    Biplab Ghosh; Anirban Sain

    2008-08-01

    During the last phase of cell division in bacteria, a polymeric ring forms at the division site. The ring, made of intracellular proteins, anchors to the cell wall and starts to contract. That initiates a dividing septum to close in, like the shutter of a camera, eventually guillotining the cell into two daughters. All through, the ring remains at the leading edge of the septum and seems to power its closure. It is not understood why does the ring contract. We propose a theoretical model to explain this. It is worth mentioning that a similar contraction phenomenon occurs for the actin ring in eukaryotes, but there it is due to motor proteins, which however, are absent in bacteria.

  19. Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes

    Directory of Open Access Journals (Sweden)

    Romeo Tony

    2009-01-01

    Full Text Available Abstract Background The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization. Results The ultrastructure of cells of four members of phylum Verrucomicrobia – Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 – was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM, as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane. Conclusion A unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two

  20. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

    DEFF Research Database (Denmark)

    Yu, Ran; Graf, Joerg; Smets, Barth F.

    2008-01-01

    Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using β......-agarase I). The results indicated that the enzymatic method was much gentler on IOB cells and yielded an approximately 5000-fold higher DNA mass than the published method. The 16S rRNA gene clone library developed from the β-agarase I treated IOB enrichments indicated a high IOB community diversity...

  1. Modeling the induced mutation process in bacterial cells with defects in excision repair system

    Science.gov (United States)

    Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2015-12-01

    A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.

  2. Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells.

    OpenAIRE

    Levis, R; Huang, H.; Schlesinger, S

    1987-01-01

    We are investigating the feasibility of using the positive-strand RNA virus Sindbis virus and its defective interfering (DI) particles as vectors for introducing foreign genes into cells. In previous work we showed by deletion mapping of a cloned cDNA derived from one of the DI RNAs that only nucleotides at the 3' and 5' termini of the RNA are essential for the DI RNA to be amplified after it is transfected into cells in the presence of helper virus. As a first step in developing a vector we ...

  3. FACTORS LIMITING BACTERIAL GROWTH : III. CELL SIZE AND "PHYSIOLOGIC YOUTH" IN BACTERIUM COLI CULTURES.

    Science.gov (United States)

    Hershey, A D; Bronfenbrenner, J

    1938-07-20

    1. Measurements of the rate of oxygen uptake per cell in transplants of Bacterium coli from cultures of this organism in different phases of growth have given results in essential agreement with the observations of others. 2. Correlations of viable count, centrifugable nitrogen, and turbidity, with oxygen consumption, indicate that the increased metabolism during the early portion of the growth period is quantitatively referable to increased average size of cells. 3. Indirect evidence has suggested that the initial rate of growth of transplants is not related to the phase of growth of the parent culture.

  4. Characterization of bacterial and archaeal communities in air-cathode microbial fuel cells, open circuit and sealed-off reactors

    KAUST Repository

    Chehab, Noura A.

    2013-06-18

    A large percentage of organic fuel consumed in a microbial fuel cell (MFC) is lost as a result of oxygen transfer through the cathode. In order to understand how this oxygen transfer affects the microbial community structure, reactors were operated in duplicate using three configurations: closed circuit (CC; with current generation), open circuit (OC; no current generation), and sealed off cathodes (SO; no current, with a solid plate placed across the cathode). Most (98 %) of the chemical oxygen demand (COD) was removed during power production in the CC reactor (maximum of 640 ± 10 mW/m 2), with a low percent of substrate converted to current (coulombic efficiency of 26.5 ± 2.1 %). Sealing the cathode reduced COD removal to 7 %, but with an open cathode, there was nearly as much COD removal by the OC reactor (94.5 %) as the CC reactor. Oxygen transfer into the reactor substantially affected the composition of the microbial communities. Based on analysis of the biofilms using 16S rRNA gene pyrosequencing, microbes most similar to Geobacter were predominant on the anodes in the CC MFC (72 % of sequences), but the most abundant bacteria were Azoarcus (42 to 47 %) in the OC reactor, and Dechloromonas (17 %) in the SO reactor. Hydrogenotrophic methanogens were most predominant, with sequences most similar to Methanobacterium in the CC and SO reactor, and Methanocorpusculum in the OC reactors. These results show that oxygen leakage through the cathode substantially alters the bacterial anode communities, and that hydrogenotrophic methanogens predominate despite high concentrations of acetate. The predominant methanogens in the CC reactor most closely resembled those in the SO reactor, demonstrating that oxygen leakage alters methanogenic as well as general bacterial communities. © 2013 Springer-Verlag Berlin Heidelberg.

  5. Capsular Sialic Acid of Streptococcus suis Serotype 2 Binds to Swine Influenza Virus and Enhances Bacterial Interactions with Virus-Infected Tracheal Epithelial Cells

    Science.gov (United States)

    Wang, Yingchao; Gagnon, Carl A.; Savard, Christian; Music, Nedzad; Srednik, Mariela; Segura, Mariela; Lachance, Claude; Bellehumeur, Christian

    2013-01-01

    Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model. PMID:24082069

  6. A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

    NARCIS (Netherlands)

    Ailor, E; Pathmanathan, J; Jongbloed, JDH; Betenbaugh, MJ

    1999-01-01

    The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the sig

  7. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  8. EzrA: a spectrin-like scaffold in the bacterial cell division machinery

    Directory of Open Access Journals (Sweden)

    Robert M Cleverley

    2015-01-01

    Full Text Available Much progress has been made in identifying the components of the divisome, the assembly of proteins that undertakes the vital process of cell division in bacteria. However, how the highly interdependent processes on either side of the membrane are coordinated during division is a major unresolved question. How is the degradation and synthesis of the cell wall on the outside of the cell coordinated with cytokinesis and membrane fission, which are driven from the inside of the cell by the tubulin homologue FtsZ? A possible key mediator of such coordination is the membrane protein EzrA, as it interacts both with FtsZ and the penicillin binding proteins (PBPs that synthesize peptidoglycan. Cleverley et al. [Nature Communications (2014 5, 5421] have recently solved the crystal structure of the cytoplasmic domain of B. subtilis EzrA, which points to an important scaffolding role for EzrA in the divisome. The structure resembles the eukaryotic, cytoskeletal spectrin proteins, which link actin filaments in the cytoskeleton and also connect the actin cytoskeleton to membrane-bound integrin proteins.

  9. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection

    NARCIS (Netherlands)

    Bartfeld, Sina; Bayram, Tülay; van de Wetering, Marc; Huch, Meritxell; Begthel, Harry; Kujala, Pekka; Vries, Robert; Peters, Peter J; Clevers, Hans

    2015-01-01

    BACKGROUND & AIMS: We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technolo

  10. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  11. Robotic milking and milk quality: effects on bacterial counts, somatic cell counts, freezing point and free fatty acids

    Directory of Open Access Journals (Sweden)

    Yvonne van der Vorst

    2010-01-01

    Full Text Available Changes in milk quality after the introduction of automatic milking systems (AM-systems on dairy farms in TheNetherlands, Germany and Denmark were examined and the data were compared with milk quality results of farms withconventional milking technology. After introduction, a small, but significant increase in total bacterial count, somatic cellcount, freezing point and free fatty acids was observed. The highest levels for total plate count and cell count are foundin the first six months after introduction. After this period the milk quality slightly improves to a more stable level.Risk factors related with milk quality concern general farm characteristics, animal health, AM-system, cleaning and cooling,housing, management skills of the farmer and the hygiene on the farm. Total plate count was significantly relatedto milk yield of the herd, cleaning of the area around the AM-system and the overall hygiene on the farm. Bulk milksomatic cell count appeared to be significantly related to milk yield of the herd and the number of milkings before replacementof the liners. An increased milking frequency is not the only explanation of increased free fatty acid levels. Technicalfactors related to free fatty acids mainly concerned the air inlet in the teat cups, bubbling (excessive air inlet and a toolong post run time of the milk pump. However, several questions regarding the causes of increased free fatty acid levelsremained unclear.

  12. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  13. The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck.

    Directory of Open Access Journals (Sweden)

    Alice Gerlini

    2014-03-01

    Full Text Available The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.

  14. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  15. Volatiles from Subtropical Convolvulaceae That Interfere with Bacterial Cell-to-Cell Communication as Potential Antipathogenic Drugs

    Directory of Open Access Journals (Sweden)

    María C. Luciardi

    2016-01-01

    Full Text Available Increasing chronic bacterial infections create an urgent need for new antimicrobial agents or strategies for their control. Targeting virulence is one of the alternative approaches to find new medicines to treat persistent infections due to bacteria with biofilm-phenotype which are more resistant to antibiotics than their planktonic counterparts having an extreme capacity for evading the host defences. A bioguided study of sixteen extracts from flowers and leaves of four subtropical Convolvulaceae species provided evidence of the occurrence of antipathogenic natural products active against Gram positive and negative bacteria. Particularly, volatile metabolites from Merremia dissecta creeper, a food and medicinal plant, were able to interfere with the Pseudomonas aeruginosa quorum sensing system by a strong decrease of N-acyl homoserine lactone (AHL biosynthesis (63–75%, which attenuated the virulence factor expression like biofilm (55% and elastase activity (up to 27%, key factors that enable the colonization and dissemination of the infection in the host. Control of the P. aeruginosa biofilm and the QS process by phytochemicals, such as (+ spathulenol, isolated from a bioactive extract of M. dissecta leaves would be a good strategy for the development of new and effective antipathogenic drugs.

  16. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

    OpenAIRE

    Atif, Shaikh M.; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L.J.; McSorley, Stephen J.

    2014-01-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we exam...

  17. Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence

    Science.gov (United States)

    Clark, Laura C.; Seipke, Ryan F.; Prieto, Pilar; Willemse, Joost; van Wezel, Gilles P.; Hutchings, Matthew I.; Hoskisson, Paul A.

    2013-01-01

    Understanding the evolution of virulence is key to appreciating the role specific loci play in pathogenicity. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find homologues of virulence loci. One example of this is the mammalian cell entry (mce) locus, which has been characterised in Mycobacterium tuberculosis. To investigate the role in Streptomyces we deleted the mce locus and studied its impact on cell survival, morphology and interaction with other soil organisms. Disruption of the mce cluster resulted in virulence towards amoebae (Acanthamoeba polyphaga) and reduced colonization of plant (Arabidopsis) models, indicating these genes may play an important role in Streptomyces survival in the environment. Our data suggest that loss of mce in Streptomyces spp. may have profound effects on survival in a competitive soil environment, and provides insight in to the evolution and selection of these genes as virulence factors in related pathogenic organisms. PMID:23346366

  18. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    Science.gov (United States)

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  19. A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

    Science.gov (United States)

    Bertolo, Lisa; Monteiro, Mario A.; Agellon, Al; Viswanathan, V. K.; Vedantam, Gayatri

    2016-01-01

    Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. PMID:27741317

  20. Impact of Alkyl Polyglucosides Surfactant Lutensol GD 70 on Modification of Bacterial Cell Surface Properties

    OpenAIRE

    Smułek, Wojciech; Kaczorek, Ewa; Zgoła-Grzeskowiak, Agnieszka; Cybulski, Zefiryn

    2015-01-01

    Alkyl polyglucosides, due to their low toxicity and environmental compatibility, could be used in biodegradation of hydrophobic compounds. In this study, the influence of Lutensol GD 70 on the cell hydrophobicity and zeta potential was measured. The particle size distribution and surfactant biodegradation were also investigated. Microbacterium sp. strain E19, Pseudomonas stutzeri strain 9, and the same strain cultivated in stress conditions were used in studies. Adding surfactant to the diese...

  1. Biosorption of heavy metal by thermotolerant polymerproducing bacterial cells and the bioflocculant

    Directory of Open Access Journals (Sweden)

    Saithong Kaewchai

    2002-07-01

    Full Text Available Three strains of thermotolerant polymer-producing bacteria; Bacillus subtilis WD 90, Bacillus subtilis SM 29, and Enterobacter agglomerans SM 38 as well as their biofloculants were used to investigate on the adsorption of heavy metal, nickel and cadmium. The effects of pH and concentrations of heavy metal were investigated. The optimum pH for nickel and cadmium adsorption by the dried cells of E. agglomerans SM 38 were found to be 7.0 (25.5% removal and 8.0 (32% removal, respectively. For B. subtilis WD 90 and B. subtilis SM 29, the optimum pH at 8.0 exhibited the nickel removal of 27% and 25%, respectively, and cadmium removal of 28% and 28.5%, respectively. The heavy metal adsorption by the dried cells and wet cells of E. agglomerans SM 38 were slightly increased with increasing initial concentrations of nickel and cadmium up to 60 and 30 ppm, respectively. The bioflocculant of B. subtilis WD 90 and B. subtilis SM 29 showed the highest nickel removal of 90.7% and 87.0% respectively, while the cadmium removal was 90.9 and 91.4%, respectively. The optimum pH for adsorption of both nickel and cadmium by the bioflocculant of E. agglomerans SM 38 was 7.0 with the removal of 92.8 and 84.2%, respectively. The optimum nickel concentration for adsorption by the bioflocculant of E. agglomerans SM 38 was 10 ppm, with the removal of 92.5%, and rather stable up to 60 ppm. The optimum cadmium concentration for adsorption by the bioflocculant of B. subtilis SM 29 was 60 ppm at pH 8.0 with the removal of 85.7%. Therefore, the bioflocculant of the three isolates gave higher heavy metal adsorption than the cells.

  2. The percentage of living bacterial cells related to organic carbon release from senescent oceanic phytoplankton

    OpenAIRE

    S. Lasternas; S. Agustí

    2014-01-01

    Bacteria recycle vast amounts of organic carbon, playing key biogeochemical and ecological roles in the ocean. Bacterioplankton dynamics are expected to be dependent on phytoplankton primary production, but there is a high diversity of processes (e.g., sloppy feeding, cell exudation, viral lysis) involved in the transfer of primary production to dissolved organic carbon available to bacteria. Here, we show the percentage of living heterotrophic bacterioplankton in the subtro...

  3. Design and construction of a whole cell bacterial 4-hydroxyphenylacetic acid and 2-phenylacetic acid bioassay

    Directory of Open Access Journals (Sweden)

    Seppe eDierckx

    2015-06-01

    Full Text Available IntroductionAuxins are hormones that regulate plant growth and development. To accurately quantify the low levels of auxins present in plant and soil samples, sensitive detection methods are needed. In this study, the design and construction of two different whole cell auxin biosensors is illustrated. Both use the auxin responsive element HpaA as an input module and differ in output module. The first biosensor is combined with the gfp gene to produce a fluorescent biosensor. Whereas the second one is combined with the genes textit and phzS to produce a pyocyanin producing biosensor whose product can be measured electrochemically.section{Results} The fluorescent biosensor is able to detect 4-hydroxyphenylacetic acid (4-HPA and 2-phenylacetic acid (PAA concentrations from 15 µM to 3 mM in a dose-responsive manner. The pyocyanin producing biosensor is able to detect 4-HPA concentrations from 1.9 µM to 15.625 µM and PAA concentrations from 15.625 µM to 125 µM, both in a dose-responsive manner.ConclusionsA fluorescent whole cell auxin biosensor and an electrochemical whole cell auxin biosensor have been constructed and tested. Both are able to detect 4-HPA and PAA concentrations that are environmentally relevant in respect to plant growth.

  4. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.

    Science.gov (United States)

    Pavlicek, Rebecca L; Fine-Coulson, Kari; Gupta, Tuhina; Quinn, Frederick D; Posey, James E; Willby, Melisa; Castro-Garza, Jorge; Karls, Russell K

    2015-12-01

    Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets.

  5. Speciation and transformations of cobalt(II) in bacterial cells using emission (57Co) Moessbauer spectroscopy

    International Nuclear Information System (INIS)

    Complete text of publication follows. The 57Co emission variant of Moessbauer spectroscopy (EMS), despite its solitary applications in biology owing to intrinsic methodological difficulties (Yu.D. Perfiliev, A.A. Kamnev, Moessbauer Effect Ref. and Data J., 30 (2007) 121-122; A.A. Kamnev, J. Mol. Struct., 744-747 (2005) 161-167), is highly sensitive and informative. The parameters of 57Co emission spectra provide chemical speciation data for the 57Co cation (chemical state, coordination environment and symmetry, etc.), as well as quantitative information on its distribution between different cation-binding sites in complicated biosystems (A.A. Kamnev, in 'Metal Ions in Biology and Medicine', Vol. 10, John Libbey Eurotext, Paris (2008), pp. 522-527). 57Co EMS can be successfully applied for monitoring 57Co2+ interactions with microbial cells, including its metabolic transformations (A.A. Kamnev et al., Anal. Chim. Acta, 573-574 (2006) 445-452). Comparative studies in rapidly frozen aqueous suspensions of live and dead cells of the ubiquitous phytostimulating soil bacterium Azospirillum brasilense have shown similarities in the chemical species formed upon purely chemical interaction of 57Co2+ traces with dead cell biomass and those formed upon primary rapid steps (2 min) of 57Co2+ sorption by the surface of live cells. For live cells, however, the parameters of 57Co emission spectra were found to change within an hour, which reflected ongoing metabolic transformations of the cation. The data obtained are in good agreement with the recently discovered involvement of Co2+ in reactions with labile [Fe-S] clusters during their de novo biosynthesis or repair in E. coli (C. Ranquet et al., J. Biol. Chem., 282 (2007) 30442-30451), presenting the molecular basis for Co2+ toxicity, besides Co2+-induced oxidative stress. The results obtained show that 57Co EMS can provide unique information both for speciation bioanalysis and for the monitoring of radionuclide bioleaching and

  6. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    Directory of Open Access Journals (Sweden)

    Maura De Simone

    Full Text Available Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s may have a role in the reduction of cell-mediated immunity playing a critical role in

  7. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    Science.gov (United States)

    De Simone, Maura; Spagnuolo, Lorenza; Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  8. Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals.

    Science.gov (United States)

    Alquier, Marjorie; Kassim, Caroline; Bertron, Alexandra; Sablayrolles, Caroline; Rafrafi, Yan; Albrecht, Achim; Erable, Benjamin

    2014-01-01

    After closure of a waste disposal cell in a repository for radioactive waste, resaturation is likely to cause the release of soluble species contained in cement and bituminous matrices, such as ionic species (nitrates, sulfates, calcium and alkaline ions, etc.), organic matter (mainly organic acids), or gases (from steel containers and reinforced concrete structures as well as from radiolysis within the waste packages). However, in the presence of nitrates in the near-field of waste, the waste cell can initiate oxidative conditions leading to enhanced mobility of redox-sensitive radionuclides (RN). In biotic conditions and in the presence of organic matter and/or hydrogen as electron donors, nitrates may be microbiologically reduced, allowing a return to reducing conditions that promote the safety of storage. Our work aims to analyze the possible microbial reactivity of nitrates at the bitumen - concrete interface in conditions as close as possible to radioactive waste storage conditions in order (i) to evaluate the nitrate reaction kinetics; (ii) to identify the by-products (NO2(-), NH4(+), N2, N2O, etc.); and (iii) to discriminate between the roles of planktonic bacteria and those adhering as a biofilm structure in the denitrifying activity. Leaching experiments on solid matrices (bitumen and cement pastes) were first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface, e.g. highly alkaline pH conditions (10 < pH < 11) imposed by the cement matrix. The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these particular conditions of pH. The denitrifying activity of H. desiderata was quantified in a batch bioreactor in the presence of solid matrices and/or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement

  9. Comparison of availability of copper(II) complexes with organic ligands to bacterial cells and to chitin

    Energy Technology Data Exchange (ETDEWEB)

    Vasconcelos, M.T.S.D.; Azenha, M.A.O. [Laquipai, Porto (Portugal). Faculdade de Ciencias do Porto; Cabral, J.P.S. [Inst. de Botanica e Centro de Citologia Experimental U.P., Porto (Portugal)

    1997-10-01

    Bacterial cells or chitin were exposed to solutions with 100 {micro}M total but only 5 {micro}M free copper, due to the presence of a proper concentration of proline, lysine, cysteine, or ethylenediamine tetraacetate (EDTA). The influence of the nature and concentration of the particles and soluble ligands, on the sorption and on the desorption of the copper, at pH 6.50 and 25.0 C, was investigated. The metal sorbed by the particles and that left in the solution were measured by atomic absorption spectrometry, after different periods of contact between particles and solution. The interpretation of the results was based on the copper(II) speciation calculated through equilibrium approaches applied to homogeneous or heterogeneous systems. A significant fraction of copper bound to the organic ligands was displaced to the bacteria or chitin, and the extent of chemical reaction depended on the nature of both the soluble (or leaving) ligands and sites on the particle surface (or entering ligands), as expected by the equilibrium theory. But with chitin, the uptake of copper in the presence of cysteine or EDTA was higher than expected, which may be due to the adsorption of the soluble copper complexes on the particle surface. In consequence of a competition between soluble and particulate ligands (cells or chitin), the free copper(II) concentration decreased in the solution, even in the presence of very strong chelators. The results indicate that copper availability is not a simple function of the initial free copper concentration in the solution. Desorption of the previously fixed copper, originated by free soluble ligands indicated that the sorption of copper was quasireversible for both particles, though a larger dismissal of the equilibrium position occurred for the cells, probably due to their biological activity.

  10. Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells

    OpenAIRE

    Jichuan Zhang; Jingyi Fei; Leslie, Benjamin J.; Kyu Young Han; Kuhlman, Thomas E; Taekjip Ha

    2015-01-01

    Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs po...

  11. Hierarchical micro/nanostructured titanium with balanced actions to bacterial and mammalian cells for dental implants

    Directory of Open Access Journals (Sweden)

    Zhu Y

    2015-10-01

    Full Text Available Yu Zhu,1,* Huiliang Cao,2,* Shichong Qiao,1,* Manle Wang,2,3 Yingxin Gu,1 Huiwen Luo,1 Fanhao Meng,2 Xuanyong Liu,2 Hongchang Lai1 1Department of Oral and Maxillofacial Implantology, Shanghai Key Laboratory of Stomatology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 3School of Materials Engineering, Shanghai University of Engineering Science, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: A versatile strategy to endow dental implants with long-term antibacterial ability without compromising the cytocompatibility is highly desirable to combat implant-related infection. Silver nanoparticles (Ag NPs have been utilized as a highly effective and broad-spectrum antibacterial agent for surface modification of biomedical devices. However, the high mobility and subsequent hazardous effects of the particles on mammalian cells may limit its practical applications. Thus, Ag NPs were immobilized on the surface of sand-blasted, large grit, and acid-etched (SLA titanium by manipulating the atomic-scale heating effect of silver plasma immersion ion implantation. The silver plasma immersion ion implantation-treated SLA surface gave rise to both good antibacterial activity and excellent compatibility with mammalian cells. The antibacterial activity rendered by the immobilized Ag NPs was assessed using Fusobacterium nucleatum and Staphylococcus aureus, commonly suspected pathogens for peri-implant disease. The immobilized Ag NPs offered a good defense against multiple cycles of bacteria attack in both F. nucleatum and S. aureus, and the mechanism was independent of silver release. F. nucleatum showed a higher susceptibility to Ag NPs than S. aureus, which might be explained by the presence of different wall structures. Moreover, the

  12. A novel receptor – ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

    Directory of Open Access Journals (Sweden)

    Charbit Alain

    2008-09-01

    Full Text Available Abstract Background Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested. Results We show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu, a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin. Discussion Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of

  13. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...

  14. RND type efflux pump system MexAB-OprM of pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication

    Directory of Open Access Journals (Sweden)

    Minagawa Shu

    2012-05-01

    Full Text Available Abstract Background Bacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs as signal compounds for quorum sensing (QS within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. Results Loss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment. Conclusion This work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P

  15. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9.

    Science.gov (United States)

    Atif, Shaikh M; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L J; McSorley, Stephen J

    2015-02-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses. PMID:25430631

  16. Depletion of T cell epitopes in lysostaphin mitigates anti-drug antibody response and enhances antibacterial efficacy in vivo

    Science.gov (United States)

    Zhao, Hongliang; Verma, Deeptak; Li, Wen; Choi, Yoonjoo; Ndong, Christian; Fiering, Steven N.; Bailey-Kellogg, Chris; Griswold, Karl E.

    2015-01-01

    SUMMARY The enzyme lysostaphin possesses potent anti-staphylococcal activity and represents a promising antibacterial drug candidate; however, its immunogenicity poses a barrier to clinical translation. Here, structure-based biomolecular design enabled widespread depletion of lysostaphin’s DRB1*0401 restricted T cell epitopes, and resulting deimmunized variants exhibited striking reductions in anti-drug antibody responses upon administration to humanized HLA-transgenic mice. This reduced immunogenicity translated into improved efficacy in the form of protection against repeated challenges with methicillin-resistant Staphylococcus aureus, or MRSA. In contrast, while wild type lysostaphin was efficacious against the initial MRSA infection, it failed to clear subsequent bacterial challenges that were coincident with escalating anti-drug antibody titers. These results extend the existing deimmunization literature, in which reduced immunogenicity and retained efficacy are assessed independently of each other. By correlating in vivo efficacy with longitudinal measures of anti-drug antibody development, we provide the first direct evidence that T cell epitope depletion manifests enhanced biotherapeutic efficacy. PMID:26000749

  17. Whole Cell Imprinting in Sol-Gel Thin Films for Bacterial Recognition in Liquids: Macromolecular Fingerprinting

    Directory of Open Access Journals (Sweden)

    Robert Armon

    2010-03-01

    Full Text Available Thin films of organically modified silica (ORMOSILS produced by a sol-gel method were imprinted with whole cells of a variety of microorganisms in order to develop an easy and specific probe to concentrate and specifically identify these microorganisms in liquids (e.g., water. Microorganisms with various morphology and outer surface components were imprinted into thin sol-gel films. Adsorption of target microorganism onto imprinted films was facilitated by these macromolecular fingerprints as revealed by various microscopical examinations (SEM, AFM, HSEM and CLSM. The imprinted films showed high selectivity toward each of test microorganisms with high adsorption affinity making them excellent candidates for rapid detection of microorganisms from liquids.

  18. Industrial Production of Therapeutic Proteins: Cell Lines, Cell Culture, and Purification

    Science.gov (United States)

    Zhu, Marie M.; Mollet, Michael; Hubert, Rene S.

    The biotechnology and pharmaceutical industries have seen a recent surge in the development of biological drug products manufactured from engineered mammalian cell lines. Since the hugely successful launch of human tissue plasminogen activator in 1987 and erythropoietin in 1988, the biopharmaceutical market has grown immensely. Global sales in 2003 exceeded US 30 billion.1 Currently, a total of 108 biotherapeutics are approved and available to patients (Table 32.1). In addition, 324 medically related, biotechnology-derived medicines for nearly 150 diseases are in clinical trials or under review by the U.S. Food and Drug Administration.2 These biopharmaceutical candidates promise to bring more and better treatments to patients. Compared to small molecule drugs, biotherapeutics show exquisite specificity with fewer off-target interactions and improved safety profiles.

  19. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); Zhang, Shu [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Lang, Qiaolin [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Song, Jianxia; Han, Lihui [Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Liu, Aihua, E-mail: liuah@qibebt.ac.cn [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China)

    2015-07-16

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP{sup +}-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP{sup +} involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  20. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    International Nuclear Information System (INIS)

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP+-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP+ involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection

  1. Characterization of the COD removal, electricity generation, and bacterial communities in microbial fuel cells treating molasses wastewater.

    Science.gov (United States)

    Lee, Yun-Yeong; Kim, Tae G; Cho, Kyung-Suk

    2016-11-01

    The chemical oxygen demand (COD) removal, electricity generation, and microbial communities were compared in 3 types of microbial fuel cells (MFCs) treating molasses wastewater. Single-chamber MFCs without and with a proton exchange membrane (PEM), and double-chamber MFC were constructed. A total of 10,000 mg L(-1) COD of molasses wastewater was continuously fed. The COD removal, electricity generation, and microbial communities in the two types of single-chamber MFCs were similar, indicating that the PEM did not enhance the reactor performance. The COD removal in the single-chamber MFCs (89-90%) was higher than that in the double-chamber MFC (50%). However, electricity generation in the double-chamber MFC was higher than that in the single-chamber MFCs. The current density (80 mA m(-2)) and power density (17 mW m(-2)) in the double-chamber MFC were 1.4- and 2.2-times higher than those in the single-chamber MFCs, respectively. The bacterial community structures in single- and double-chamber MFCs were also distinguishable. The amount of Proteobacteria in the double-chamber MFC was 2-3 times higher than those in the single-chamber MFCs. For the archaeal community, Methanothrix (96.4%) was remarkably dominant in the single-chamber MFCs, but Methanobacterium (35.1%), Methanosarcina (28.3%), and Methanothrix (16.2%) were abundant in the double-chamber MFC.

  2. The Use of MALDI-TOF-MS and In Silico Studies for Determination of Antimicrobial Peptides' Affinity to Bacterial Cells

    Science.gov (United States)

    Mandal, Santi M.; Migliolo, Ludovico; Franco, Octavio L.

    2012-11-01

    Several methods have been proposed for determining the binding affinity of antimicrobial peptides (AMPs) to bacterial cells. Here the utilization of MALDI-TOF-MS was proposed as a reliable and efficient method for high throughput AMP screening. The major advantage of the technique consists of finding AMPs that are selective and specific to a wide range of Gram-negative and -positive bacteria, providing a simple reliable screening tool to determine the potential candidates for broad spectrum antimicrobial drugs. As a prototype, amp-1 and -2 were used, showing highest activity toward Gram-negative and -positive membranes respectively. In addition, in silico molecular docking studies with both peptides were carried out for the membranes. In silico results indicated that both peptides presented affinity for DPPG and DPPE phospholipids, constructed in order to emulate an in vivo membrane bilayer. As a result, amp-1 showed a higher complementary surface for Gram-negative while amp-2 showed higher affinity to Gram-positive membranes, corroborating MS analyses. In summary, results here obtained suggested that in vitro methodology using MALDI-TOF-MS in addition to theoretical studies may be able to improve AMP screening quality.

  3. Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

    Indian Academy of Sciences (India)

    Arnab Roy; Umesh Varshney; Debnath Pal

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  4. Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae.

    Science.gov (United States)

    Schröder, Gunnar; Schuelein, Ralf; Quebatte, Maxime; Dehio, Christoph

    2011-08-30

    Bacterial type IV secretion systems (T4SS) mediate interbacterial conjugative DNA transfer and transkingdom protein transfer into eukaryotic host cells in bacterial pathogenesis. The sole bacterium known to naturally transfer DNA into eukaryotic host cells via a T4SS is the plant pathogen Agrobacterium tumefaciens. Here we demonstrate T4SS-mediated DNA transfer from a human bacterial pathogen into human cells. We show that the zoonotic pathogen Bartonella henselae can transfer a cryptic plasmid occurring in the bartonellae into the human endothelial cell line EA.hy926 via its T4SS VirB/VirD4. DNA transfer into EA.hy926 cells was demonstrated by using a reporter derivative of this Bartonella-specific mobilizable plasmid generated by insertion of a eukaryotic egfp-expression cassette. Fusion of the C-terminal secretion signal of the endogenous VirB/VirD4 protein substrate BepD with the plasmid-encoded DNA-transport protein Mob resulted in a 100-fold increased DNA transfer rate. Expression of the delivered egfp gene in EA.hy926 cells required cell division, suggesting that nuclear envelope breakdown may facilitate passive entry of the transferred ssDNA into the nucleus as prerequisite for complementary strand synthesis and transcription of the egfp gene. Addition of an eukaryotic neomycin phosphotransferase expression cassette to the reporter plasmid facilitated selection of stable transgenic EA.hy926 cell lines that display chromosomal integration of the transferred plasmid DNA. Our data suggest that T4SS-dependent DNA transfer into host cells may occur naturally during human infection with Bartonella and that these chronically infecting pathogens have potential for the engineering of in vivo gene-delivery vectors with applications in DNA vaccination and therapeutic gene therapy. PMID:21844337

  5. Application of quartz tuning forks for detection of endotoxins and Gram-negative bacterial cells by monitoring of Limulus Amebocyte Lysate coagulation.

    Science.gov (United States)

    Chałupniak, Andrzej; Waszczuk, Karol; Hałubek-Głuchowska, Katarzyna; Piasecki, Tomasz; Gotszalk, Teodor; Rybka, Jacek

    2014-08-15

    Endotoxins, pyrogens of bacterial origin, are a significant threat in many areas of life. Currently, the test most commonly used for endotoxin level determination is LAL (Limulus Amebocyte Lysate) assay. This paper presents application of commercially available low-frequency piezoelectric tuning forks (QTFs) for endotoxin detection. Measurement of the decrease in the QTF oscillation amplitude provides information about the viscosity changes, occurring in the tested sample upon addition of LAL. That method was used to determine the concentrations of endotoxins and bacterial cells (E. coli O157:H19). The relevance of the obtained results was confirmed using a commercially available colorimetric LAL assay. The constructed system can detect bacterial endotoxins in the range of 0.001-5EU/ml and bacterial cells in the range of 10(2)-10(7)CFU/ml. The presented technique requires very simple sample preparation and the sensor response is obtained using compact, portable readout electronics. The single test cost is low compared to commercial endotoxin assays and other novel systems based on micromechanical sensors. PMID:24632139

  6. Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

    OpenAIRE

    Deng, Meihong; Scott, Melanie J.; Loughran, Patricia; Gibson, Gregory; Sodhi, Chhinder; Watkins, Simon; Hackam, David; Billiar, Timothy R

    2013-01-01

    The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then...

  7. A novel approach to recycle bacterial culture waste for fermentation reuse via a microbial fuel cell-membrane bioreactor system.

    Science.gov (United States)

    Li, Jian; Zhu, Yuan; Zhuang, Liangpeng; Otsuka, Yuichiro; Nakamura, Masaya; Goodell, Barry; Sonoki, Tomonori; He, Zhen

    2015-09-01

    Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.

  8. Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

    Science.gov (United States)

    Shpigel, Etai; Roiz, Levava; Goren, Raphael; Shoseyov, Oded

    1998-01-01

    Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. PMID:9701575

  9. Computing with bacterial constituents, cells and populations: from bioputing to bactoputing.

    Science.gov (United States)

    Norris, Vic; Zemirline, Abdallah; Amar, Patrick; Audinot, Jean Nicolas; Ballet, Pascal; Ben-Jacob, Eshel; Bernot, Gilles; Beslon, Guillaume; Cabin, Armelle; Fanchon, Eric; Giavitto, Jean-Louis; Glade, Nicolas; Greussay, Patrick; Grondin, Yohann; Foster, James A; Hutzler, Guillaume; Jost, Jürgen; Kepes, Francois; Michel, Olivier; Molina, Franck; Signorini, Jacqueline; Stano, Pasquale; Thierry, Alain R

    2011-09-01

    The relevance of biological materials and processes to computing-alias bioputing-has been explored for decades. These materials include DNA, RNA and proteins, while the processes include transcription, translation, signal transduction and regulation. Recently, the use of bacteria themselves as living computers has been explored but this use generally falls within the classical paradigm of computing. Computer scientists, however, have a variety of problems to which they seek solutions, while microbiologists are having new insights into the problems bacteria are solving and how they are solving them. Here, we envisage that bacteria might be used for new sorts of computing. These could be based on the capacity of bacteria to grow, move and adapt to a myriad different fickle environments both as individuals and as populations of bacteria plus bacteriophage. New principles might be based on the way that bacteria explore phenotype space via hyperstructure dynamics and the fundamental nature of the cell cycle. This computing might even extend to developing a high level language appropriate to using populations of bacteria and bacteriophage. Here, we offer a speculative tour of what we term bactoputing, namely the use of the natural behaviour of bacteria for calculating. PMID:21384168

  10. Link between Domoic Acid Production and Cell Physiology after Exchange of Bacterial Communities between Toxic Pseudo-nitzschia multiseries and Non-Toxic Pseudo-nitzschia delicatissima

    Directory of Open Access Journals (Sweden)

    Aurélie Lelong

    2014-06-01

    Full Text Available Bacteria are known to influence domoic acid (DA production by Pseudo-nitzschia spp., but the link between DA production and physiology of diatoms requires more investigation. We compared a toxic P. multiseries to a non-toxic P. delicatissima, investigating links between DA production, physiological parameters, and co-occurring bacteria. Bacterial communities in cultures of both species were reduced by antibiotic treatment, and each of the diatoms was inoculated with the bacterial community of the other species. The physiology of P. delicatissima was minimally affected by the absence of bacteria or the presence of alien bacteria, and no DA was detected. P. multiseries grew faster without bacteria, did not produce a significant amount of DA, and exhibited physiological characteristics of healthy cells. When grown with alien bacteria, P. multiseries did not grow and produced more DA; the physiology of these cells was affected, with decreases in chlorophyll content and photosynthetic efficiency, an increase in esterase activity, and almost 50% mortality of the cells. The alien bacterial community had morphological and cellular characteristics very different from the original bacteria, and the number of free-living bacteria per algal cell was much higher, suggesting the involvement of bacteria in DA production.

  11. Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

    OpenAIRE

    Wang, Yuanmin; Sevinc, Papatya C.; Balchik, Sara M.; Fridrickson, Jim; Shi, Liang; Lu, H. Peter

    2013-01-01

    We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles ads...

  12. Bacterial surface adaptation

    Science.gov (United States)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  13. White blood cell count, absolute neutrophil count, as predictors of hidden bacterial infections in febrile children 1-18 months of age without focus

    International Nuclear Information System (INIS)

    Objectives: To study the relationship between White Blood Cell (WBC), Absolute Neutrophil Count (ANC) in febrile children 1-18 months of age as predictor of bacterial infection, so as to improve our predictability of bacterial infections in emergency room to decrease unnecessary admissions and antibiotic use. Methods: Retrospective review was performed on febrile patients 1-18 months of age that were admitted to hospital between August 2002 and March 2003 on the presumptive diagnosis of fever without focus, Complete septic work up was done for all patients according to local hospital protocol including Complete blood count (CBC), blood culture, urine culture, Chest X-Ray (CXR) and lumbar puncture, Patients who had history of antibiotics use within 48 hours of admission were excluded from the study, History, physical examination, laboratory and radiology data were reviewed. Data about the age, sex, temperature, presence or absence of focal bacterial infection, WBC, ANC, CXR report and body fluid culture results were collected and analyzed. Results: Thirty-four patients were reviewed in this study, Eight patients (23.5%) had bacterial infection: classified as group (2 patchy pneumonia, 3 Urinary tract infection (UTI), 2 meningitis, 1 Occult bacteremia (OB) and 26 patients (76.5%) had no evidence of bacterial infection, classified as group 2, No significant difference was found between the two groups in respect to age, sex, temperature and WBC P>0.05, while there was a significant difference between the two groups in respect to the ANC P = 0.02, also ANC had better sensitivity (78%) and specificity (89%) than WBC (sensitivity 77%, specificity 62%). Conclusion: ANC is a good predictive test for determining bacterial infection in young febrile children without focus, However there is need for other more reliable rapid cost effective measures in dealing with young febrile children at emergency department. (author)

  14. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    aggregation and/or mislfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  15. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    /or misfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  16. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression....... jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  17. A Serine-Threonine Kinase (StkP Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro.

    Directory of Open Access Journals (Sweden)

    Jenny A Herbert

    Full Text Available The pneumococcal serine threonine protein kinase (StkP acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.

  18. Anti-infective activities of lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Servin, Alain L

    2014-04-01

    A vast and diverse array of microbial species displaying great phylogenic, genomic, and metabolic diversity have colonized the gastrointestinal tract. Resident microbes play a beneficial role by regulating the intestinal immune system, stimulating the maturation of host tissues, and playing a variety of roles in nutrition and in host resistance to gastric and enteric bacterial pathogens. The mechanisms by which the resident microbial species combat gastrointestinal pathogens are complex and include competitive metabolic interactions and the production of antimicrobial molecules. The human intestinal microbiota is a source from which Lactobacillus probiotic strains have often been isolated. Only six probiotic Lactobacillus strains isolated from human intestinal microbiota, i.e., L. rhamnosus GG, L. casei Shirota YIT9029, L. casei DN-114 001, L. johnsonii NCC 533, L. acidophilus LB, and L. reuteri DSM 17938, have been well characterized with regard to their potential antimicrobial effects against the major gastric and enteric bacterial pathogens and rotavirus. In this review, we describe the current knowledge concerning the experimental antibacterial activities, including antibiotic-like and cell-regulating activities, and therapeutic effects demonstrated in well-conducted, placebo-controlled, randomized clinical trials of these probiotic Lactobacillus strains. What is known about the antimicrobial activities supported by the molecules secreted by such probiotic Lactobacillus strains suggests that they constitute a promising new source for the development of innovative anti-infectious agents that act luminally and intracellularly in the gastrointestinal tract.

  19. Cell-based delivery of oncolytic viruses: a new strategic alliance for a biological strike against cancer.

    Science.gov (United States)

    Power, Anthony T; Bell, John C

    2007-04-01

    Recent years have seen tremendous advances in the development of exquisitely targeted replicating virotherapeutics that can safely destroy malignant cells. Despite this promise, clinical advancement of this powerful and unique approach has been hindered by vulnerability to host defenses and inefficient systemic delivery. However, it now appears that delivery of oncolytic viruses within carrier cells may offer one solution to this critical problem. In this review, we compare the advantages and limitations of the numerous cell lineages that have been investigated as delivery platforms for viral therapeutics, and discuss examples showing how combined cell-virus biotherapeutics can be used to achieve synergistic gains in antitumor activity. Finally, we highlight avenues for future preclinical research that might be taken in order to refine cell-virus biotherapeutics in preparation for human trials. PMID:17264852

  20. tmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell

    Directory of Open Access Journals (Sweden)

    Hyouta eHimeno

    2014-04-01

    Full Text Available tmRNA (transfer messenger RNA; also known as 10Sa RNA or SsrA RNA is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of nonfunctional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.

  1. Antimicrobials for bacterial bioterrorism agents.

    Science.gov (United States)

    Sarkar-Tyson, Mitali; Atkins, Helen S

    2011-06-01

    The limitations of current antimicrobials for highly virulent pathogens considered as potential bioterrorism agents drives the requirement for new antimicrobials that are suitable for use in populations in the event of a deliberate release. Strategies targeting bacterial virulence offer the potential for new countermeasures to combat bacterial bioterrorism agents, including those active against a broad spectrum of pathogens. Although early in the development of antivirulence approaches, inhibitors of bacterial type III secretion systems and cell division mechanisms show promise for the future.

  2. Bacterial wall products induce downregulation of vascular endothelial growth factor receptors on endothelial cells via a CD14-dependent mechanism: implications for surgical wound healing.

    LENUS (Irish Health Repository)

    Power, C

    2012-02-03

    INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent mitogenic cytokine which has been identified as the principal polypeptide growth factor influencing endothelial cell (EC) migration and proliferation. Ordered progression of these two processes is an absolute prerequisite for initiating and maintaining the proliferative phase of wound healing. The response of ECs to circulating VEGF is determined by, and directly proportional to, the functional expression of VEGF receptors (KDR\\/Flt-1) on the EC surface membrane. Systemic sepsis and wound contamination due to bacterial infection are associated with significant retardation of the proliferative phase of wound repair. The effects of the Gram-negative bacterial wall components lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) on VEGF receptor function and expression are unknown and may represent an important biological mechanism predisposing to delayed wound healing in the presence of localized or systemic sepsis. MATERIALS AND METHODS: We designed a series of in vitro experiments investigating this phenomenon and its potential implications for infective wound repair. VEGF receptor density on ECs in the presence of LPS and BLP was assessed using flow cytometry. These parameters were assessed in hypoxic conditions as well as in normoxia. The contribution of CD14 was evaluated using recombinant human (rh) CD14. EC proliferation in response to VEGF was quantified in the presence and absence of LPS and BLP. RESULTS: Flow cytometric analysis revealed that LPS and BLP have profoundly repressive effects on VEGF receptor density in normoxic and, more pertinently, hypoxic conditions. The observed downregulation of constitutive and inducible VEGF receptor expression on ECs was not due to any directly cytotoxic effect of LPS and BLP on ECs, as measured by cell viability and apoptosis assays. We identified a pivotal role for soluble\\/serum CD14, a highly specific bacterial wall product receptor, in

  3. SGLT1 activity in lung alveolar cells of diabetic rats modulates airway surface liquid glucose concentration and bacterial proliferation

    OpenAIRE

    Tales Lyra Oliveira; Návylla Candeia-Medeiros; Polliane M. Cavalcante-Araújo; Igor Santana Melo; Elaine Fávaro-Pípi; Luciana Alves Fátima; Antônio Augusto Rocha; Luiz Ricardo Goulart; Ubiratan Fabres Machado; Ruy R. Campos; Robinson Sabino-Silva

    2016-01-01

    High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-...

  4. Comparison of Ground Water Bacterial Cell Sizes from the Agricultural,Domestic and Industrial Areas of Mysore District,Karnataka State,India

    Institute of Scientific and Technical Information of China (English)

    Wadie Ahmed Mokbel; Sadanand M Yamakanamardi

    2008-01-01

    A two-year study on temporal variations in the ground water heterotrophic bacterial cell sizes of free living bacteria(FLB)and particle bound bacteria(PBB)from the agricultural,domestic and industrial areas was carried out from Februar y2005 to January 2007.The overall mean cell length of FLB and PBB was similar in all the ground water studied.However,the season wise grouped data revealed significant seasonal changes in cell length of FLB and PBB,as smaller bacteria were noticed during rainy season in the ground water in agricultural area in both the years,and only in the second year of study in domestic and industrial areas.Generally,it was noticed that there were summer maximum and rainy minimum values of the cell length of PBB in the ground water in agricultural,domestic and industrial areas in the second year of study.The Pearson's correlations showed the presence of 8(in agricultural area),5(in domestic)and 3(in industrial) significant correlations with environmental(Physico-chemical)parameters,respectively.The regression analysis revealed that as much as 12%of variation in the mean length of FL Bwas due to NO3(+)in agricultural area and 9%due to total solids(+)indomestic area.However,the 8% variation in bacterial cell size of FLB was due to Mg(+)in industrial area.Whereas,13%variation in mean length of PBB was due to S04(+)in agncultural area and 10%due to total anions of strong acid(TASA)(+)in domestic area.Furthermore,10% of variation Was due to PO4(+)in industrial area.Thus,the statistical analysis revealed that several environmental variables were potentially responsible for some of the temporal variations in aquatic heterotrophic bacterial cell size,suggesting probably the stressed environment in these ecosystems.

  5. Bacterial Vaginosis

    Science.gov (United States)

    ... 586. Related Content STDs during Pregnancy Fact Sheet Pregnancy and HIV, Viral Hepatitis, and STD Prevention Pelvic Inflammatory Disease ( ... Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ... STDs See Also Pregnancy Reproductive ...

  6. Bacterial Meningitis

    Science.gov (United States)

    ... Schedules Preteen & Teen Vaccines Meningococcal Disease Sepsis Bacterial Meningitis Recommend on Facebook Tweet Share Compartir On this ... serious disease. Laboratory Methods for the Diagnosis of Meningitis This manual summarizes laboratory methods used to isolate, ...

  7. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas

    2010-01-01

    A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene...... as Candidatus Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells....

  8. Adherence of Escherichia coli O157:H7 to epithelial cells in vitro and in pig gut loops is affected by bacterial culture conditions

    OpenAIRE

    Yin, Xianhua; Feng, Yanni; Wheatcroft, Roger; Chambers, James; Gong, Joshua; Gyles, Carlton L.

    2011-01-01

    The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from s...

  9. Polysaccharide-capped silver Nanoparticles inhibit biofilm formation and eliminate multi-drug-resistant bacteria by disrupting bacterial cytoskeleton with reduced cytotoxicity towards mammalian cells

    Science.gov (United States)

    Sanyasi, Sridhar; Majhi, Rakesh Kumar; Kumar, Satish; Mishra, Mitali; Ghosh, Arnab; Suar, Mrutyunjay; Satyam, Parlapalli Venkata; Mohapatra, Harapriya; Goswami, Chandan; Goswami, Luna

    2016-04-01

    Development of effective anti-microbial therapeutics has been hindered by the emergence of bacterial strains with multi-drug resistance and biofilm formation capabilities. In this article, we report an efficient green synthesis of silver nanoparticle (AgNP) by in situ reduction and capping with a semi-synthetic polysaccharide-based biopolymer (carboxymethyl tamarind polysaccharide). The CMT-capped AgNPs were characterized by UV, DLS, FE-SEM, EDX and HR-TEM. These AgNPs have average particle size of ~20–40 nm, and show long time stability, indicated by their unchanged SPR and Zeta-potential values. These AgNPs inhibit growth and biofilm formation of both Gram positive (B. subtilis) and Gram negative (E. coli and Salmonella typhimurium) bacterial strains even at concentrations much lower than the minimum inhibitory concentration (MIC) breakpoints of antibiotics, but show reduced or no cytotoxicity against mammalian cells. These AgNPs alter expression and positioning of bacterial cytoskeletal proteins FtsZ and FtsA. CMT-capped AgNPs can effectively block growth of several clinical isolates and MDR strains representing different genera and resistant towards multiple antibiotics belonging to different classes. We propose that the CMT-capped AgNPs can have potential bio-medical application against multi-drug-resistant microbes with minimal cytotoxicity towards mammalian cells.

  10. SGLT1 activity in lung alveolar cells of diabetic rats modulates airway surface liquid glucose concentration and bacterial proliferation

    Science.gov (United States)

    Oliveira, Tales Lyra; Candeia-Medeiros, Návylla; Cavalcante-Araújo, Polliane M.; Melo, Igor Santana; Fávaro-Pípi, Elaine; Fátima, Luciana Alves; Rocha, Antônio Augusto; Goulart, Luiz Ricardo; Machado, Ubiratan Fabres; Campos, Ruy R.; Sabino-Silva, Robinson

    2016-01-01

    High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it. PMID:26902517

  11. Factors influencing variation of bulk milk antibiotic residue occurrence, somatic cell count, and total bacterial count in dairy sheep flocks.

    Science.gov (United States)

    Gonzalo, C; Carriedo, J A; García-Jimeno, M C; Pérez-Bilbao, M; de la Fuente, L F

    2010-04-01

    To study the variations of bulk tank milk variables in dairy ewe flocks and to identify the main target practices and flock groups to improve milk quality and safety, a total of 71,228 records of antibiotic residue (AR) and milk yield and 68,781 records of somatic cell count (SCC) and total bacterial count (TBC) were obtained over 5 yr from the same 209 dairy ewe flocks of the Assaf breed belonging to the Consortium for Ovine Promotion of Castilla-León (Spain). Based on a logistic regression model, year, month, semester, SCC, TBC, dry therapy, and milk yield significantly contributed to AR variation. High SCC was associated with increased AR violations. When antibiotic dry therapy was implemented, AR occurrence was higher than when this practice was not used. A polynomial monthly distribution throughout the year was observed for AR occurrence; the highest values were in autumn, coinciding with low milk yields per flock. Yearly occurrences drastically diminished from 2004 (1.36%) to 2008 (0.30%), probably as a result of effective educational programs. The mixed-model ANOVA of factors influencing variation in SCC and TBC indicated that year, month, AR, dry therapy group, milking type, and year interactions were significant variation factors for SCC and TBC; mathematical model accounted for 74.1 and 35.4% of total variance for each variable, respectively. Differences in management and hygiene practice caused significant SCC and TBC variations among flocks and within flocks throughout the 5-yr study. Over time, continuously dry treated flocks showed lower logSCC (5.80) and logTBC (4.92) than untreated (6.10 and 5.18, respectively) or discontinuously dry treated (6.01 and 5.05, respectively) flocks. Continuously dry treated flocks had lower AR occurrences than did discontinuously dry treated flocks. As a whole, AR occurrence and SCC and TBC bulk tank milk variables can be used for monitoring mammary health and milk hygiene and safety in dairy sheep throughout time.

  12. Bacterial glycosyltransferase toxins.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

  13. Robust vaginal colonization of macaques with a novel vaginally disintegrating tablet containing a live biotherapeutic product to prevent HIV infection in women.

    Directory of Open Access Journals (Sweden)

    Laurel A Lagenaur

    Full Text Available MucoCept is a biotherapeutic for prevention of HIV-1 infection in women and contains a human, vaginal Lactobacillus jensenii that has been genetically enhanced to express the HIV-1 entry inhibitor, modified cyanovirin-N (mCV-N. The objective of this study was to develop a solid vaginal dosage form that supports sustained vaginal colonization of the MucoCept Lactobacillus at levels previously shown, with freshly prepared cultures, to protect macaques from SHIV infection and to test this formulation in a macaque vaginal colonization model. Vaginally disintegrating tablets were prepared by lyophilizing the formulated bacteria in tablet-shaped molds, then packaging in foil pouches with desiccant. Disintegration time, potency and stability of the tablets were assessed. For colonization, non-synchronized macaques were dosed vaginally with either one tablet or five tablets delivered over five days. Vaginal samples were obtained at three, 14, and 21 days post-dosing and cultured to determine Lactobacillus colonization levels. To confirm identity of the MucoCept Lactobacillus strain, genomic DNA was extracted from samples on days 14 and 21 and a strain-specific PCR was performed. Supernatants from bacteria were tested for the presence of the mCV-N protein by Western blot. The tablets were easy to handle, disintegrated within two minutes, potent (5.7x1011 CFU/g, and stable at 4°C and 25°C. Vaginal administration of the tablets to macaques resulted in colonization of the MucoCept Lactobacillus in 66% of macaques at 14 days post-dosing and 83% after 21 days. There was no significant difference in colonization levels for the one or five tablet dosing regimens (p=0.88 Day 14, p=0.99 Day 21. Strain-specific PCR confirmed the presence of the bacteria even in culture-negative macaques. Finally, the presence of mCV-N protein was confirmed by Western blot analysis using a specific anti-mCV-N antibody.

  14. Short communication: Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count.

    Science.gov (United States)

    Koop, G; Dik, N; Nielen, M; Lipman, L J A

    2010-06-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms, 3 bulk milk samples were collected at intervals of 2 wk. The samples were cultured for SPC, coliform count, and staphylococcal count and for the presence of Staphylococcus aureus. Furthermore, SCC (Fossomatic 5000, Foss, Hillerød, Denmark) and TBC (BactoScan FC 150, Foss) were measured. Staphylococcal count was correlated to SCC (r=0.40), TBC (r=0.51), and SPC (r=0.53). Coliform count was correlated to TBC (r=0.33), but not to any of the other variables. Staphylococcus aureus did not correlate to SCC. The contribution of the staphylococcal count to the SPC was 31%, whereas the coliform count comprised only 1% of the SPC. The agreement of the repeated measurements was low. This study indicates that staphylococci in goat bulk milk are related to SCC and make a significant contribution to SPC. Because of the high variation in bacterial counts, repeated sampling is necessary to draw valid conclusions from bulk milk culturing.

  15. The bacterial lipocalins.

    Science.gov (United States)

    Bishop, R E

    2000-10-18

    The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.

  16. Effects of pulling forces, osmotic pressure, condensing agents and viscosity on the thermodynamics and kinetics of DNA ejection from bacteriophages to bacterial cells: a computational study

    Science.gov (United States)

    Petrov, Anton S.; Douglas, Scott S.; Harvey, Stephen C.

    2013-03-01

    In this work, we report on simulations of double-stranded DNA (dsDNA) ejection from bacteriophage ϕ29 into a bacterial cell. The ejection was studied with a coarse-grained model, in which viral dsDNA was represented by beads on a torsion-less string. The bacteriophage’s capsid and the bacterial cell were defined by sets of spherical constraints. To account for the effects of the viscous medium inside the bacterial cell, the simulations were carried out using a Langevin dynamics protocol. Our simplest simulations (involving constant viscosity and no external biasing forces) produced results compatible with the push-pull model of DNA ejection, with an ejection rate significantly higher in the first part of ejection than in the latter parts. Additionally, we performed more complicated simulations, in which we included additional factors such as external forces, osmotic pressure, condensing agents and ejection-dependent viscosity. The effects of these factors (independently and in combination) on the thermodynamics and kinetics of DNA ejection were studied. We found that, in general, the dependence of ejection forces and ejection rates on the amount of DNA ejected becomes more complex if the ejection is modeled with a broader, more realistic set of parameters and influences (such as variation in the solvent’s viscosity and the application of an external force). However, certain combinations of factors and numerical parameters led to the opposition of some ejection-driving and ejection-inhibiting influences, ultimately causing an apparent simplification of the ejection profiles.

  17. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  18. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, mea

  19. Bacterial biofilms: prokaryotic adventures in multicellularity

    DEFF Research Database (Denmark)

    Webb, J.S.; Givskov, Michael Christian; Kjelleberg, S.

    2003-01-01

    The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight...... into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity....

  20. Trafficking Microenvironmental pHs of Polycationic Gene Vectors in Drug-Sensitive and Multidrug-Resistant MCF7 Breast Cancer Cells

    OpenAIRE

    Kang, Han Chang; Samsonova, Olga; Bae, You Han

    2010-01-01

    While multidrug resistance (MDR) has been a significant issue in cancer chemotherapy, delivery resistance to various anticancer biotherapeutics, including genes, has not been widely recognized as a property of MDR. This study aims to provide a better understanding of the transfection characteristics of drug-sensitive and drug-resistant cells by tracing microenvironmental pHs of two representative polymer vectors: poly(l-lysine) and polyethyleneimine. Drug-sensitive breast MCF7 cells had four-...

  1. Experimental and In Silico Modelling Analyses of the Gene Expression Pathway for Recombinant Antibody and By-Product Production in NS0 Cell Lines

    OpenAIRE

    Emma J Mead; Lesley M Chiverton; Sarah K Spurgeon; Martin, Elaine B.; Montague, Gary A.; C Mark Smales; Tobias von der Haar

    2012-01-01

    Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often va...

  2. Screening and identification of a novel target specific for hepatoma cell line HepG2 from the FliTrx bacterial peptide library

    Institute of Scientific and Technical Information of China (English)

    Wenhan Li; Ping Lei; Bing Yu; Sha Wu; Jilin Peng; Xiaoping Zhao; Huffen Zhu; Michael Kirschfink; Guanxin Shen

    2008-01-01

    To explore new targets for hepatoma research, we used a surface display library to screen novel tumor cell-specific peptides. The bacterial FliTrx system was screened with living normal liver cell line L02 and hepatoma cell line HepG2 successively to search for hepatoma-specific peptides. Three clones (Hep1, Hep2, and Hep3) were identified to be specific to HepG2 compared with L02 and other cancer cell lines.Three-dimensional structural prediction proved that peptides inserted into the active site of Escherichia coli thioredoxin (TrxA) formed certain loop structures protruding out of the surface. Western blot analysis showed that FliC/TrxA-pepfide fusion proteins could be directly used to detect HepG2 cells.Three different FliC/TrxA-peptide fusion proteins targeted the same molecule, at approximately 140 kDa, on HepG2 cells.This work presented for the first time the application of the FliTrx library in screening living cells. Three peptides were obtained that could be potential candidates for targeted liver cancer therapy.

  3. Complete Genome Sequence of Cell Culture-Attenuated Guinea Pig Cytomegalovirus Cloned as an Infectious Bacterial Artificial Chromosome

    OpenAIRE

    Yang, Dongmei; Alam, Zohaib; Cui, Xiaohong; Chen, Michael; Sherrod, Carly J.; McVoy, Michael A.; Schleiss, Mark R.; Dittmer, Dirk P

    2014-01-01

    The complete genome sequence of attenuated guinea pig cytomegalovirus cloned as bacterial artificial chromosome N13R10 was determined. Comparison to pathogenic salivary gland-derived virus revealed 13 differences, 1 of which disrupted overlapping open reading frames encoding GP129 and GP130. Attenuation of N13R10 may arise from an inability to express GP129 and/or GP130.

  4. Sensing the Structural Differences in Cellulose from Apple and Bacterial Cell Wall Materials by Raman and FT-IR Spectroscopy

    OpenAIRE

    Artur Zdunek; Monika Szymańska-Chargot; Justyna Cybulska

    2011-01-01

    Raman and Fourier Transform Infrared (FT-IR) spectroscopy was used for assessment of structural differences of celluloses of various origins. Investigated celluloses were: bacterial celluloses cultured in presence of pectin and/or xyloglucan, as well as commercial celluloses and cellulose extracted from apple parenchyma. FT-IR spectra were used to estimate of the Iβ content, whereas Raman spectra were used to evaluate the degree of crystallinity of the cellulose. The crystallinity index (XC R...

  5. Whole-cell bacterial biosensors for rapid and effective monitoring of heavy metals and inorganic pollutants in wastewater.

    Science.gov (United States)

    Olaniran, Ademola O; Hiralal, Lettisha; Pillay, Balakrishna

    2011-10-01

    The increasing number of potentially harmful pollutants in the wastewater effluent discharge necessitates the need for the development of fast and cost effective analytical techniques for extensive monitoring programmes to assess the effectiveness of the treatment process. This study compared the use of bacterial biosensors to the conventional Daphnia magna assay, Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) tests as well as chemical analysis, for monitoring the toxicity of wastewater. The bacterial biosensors constructed in this study, using S. sonnei and E. coli, were found to be sensitive to the toxicity of the wastewater effluents. A linear increase in bioluminescence with increasing concentration of heavy metals and inorganic pollutants in water was observed, with a correlation coefficient (r(2)) as high as 0.995 and 0.997, respectively. No notable correlation between biosensor toxicity and BOD and COD test results was observed. These bacterial biosensors could provide appropriate alternatives for a rapid, sensitive and cost effective detection of wastewater quality. However, the differences in sensitivity obtained for the different systems suggest that the use of a battery of toxicity assays may be required to provide a real ecotoxicological assessment of wastewater samples. PMID:21904738

  6. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...... biogeochemical processes are carried exclusively by bacteria. * Bacteria play an important role in all types of habitats including some that cannot support eukaryotic life....

  7. [Bacterial vaginosis].

    Science.gov (United States)

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  8. BTLA interaction with HVEM expressed on CD8(+ T cells promotes survival and memory generation in response to a bacterial infection.

    Directory of Open Access Journals (Sweden)

    Marcos W Steinberg

    Full Text Available The B and T lymphocyte attenuator (BTLA is an Ig super family member that binds to the herpes virus entry mediator (HVEM, a TNF receptor super family (TNFRSF member. Engagement of BTLA by HVEM triggers inhibitory signals, although recent evidence indicates that BTLA also may act as an activating ligand for HVEM. In this study, we reveal a novel role for the BTLA-HVEM pathway in promoting the survival of activated CD8(+ T cells in the response to an oral microbial infection. Our data show that both BTLA- and HVEM-deficient mice infected with Listeria monocytogenes had significantly reduced numbers of primary effector and memory CD8(+ T cells, despite normal proliferation and expansion compared to controls. In addition, blockade of the BTLA-HVEM interaction early in the response led to significantly reduced numbers of antigen-specific CD8(+ T cells. HVEM expression on the CD8(+ T cells as well as BTLA expression on a cell type other than CD8(+ T lymphocytes, was required. Collectively, our data demonstrate that the function of the BTLA-HVEM pathway is not limited to inhibitory signaling in T lymphocytes, and instead, that BTLA can provide crucial, HVEM-dependent signals that promote survival of antigen activated CD8(+ T cell during bacterial infection.

  9. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  10. Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

    Science.gov (United States)

    Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

    1985-01-01

    Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

  11. Supramolecular bacterial systems

    OpenAIRE

    Sankaran, Shrikrishnan

    2015-01-01

    For nearly over a decade, a wide variety of dynamic and responsive supramolecular architectures have been investigated and developed to address biological systems. Since the non-covalent interactions between individual molecular components in such architectures are similar to the interactions found in living systems, it was possible to integrate chemically-synthesized and naturally-occurring components to create platforms with interesting bioactive properties. Bacterial cells and recombinant ...

  12. Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana Venom with Platelets, Bacterial and Cancer Cells

    Directory of Open Access Journals (Sweden)

    Jüri Siigur

    2013-01-01

    Full Text Available Secretory phospholipasesA2 (sPLA2s form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA2s from Vipera lebetina (VLPLA2, Vipera berus berus (VBBPLA2, and Naja naja oxiana (NNOPLA2 venoms on (i human platelets, (ii four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis and (iii five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10 in vitro. sPLA2s inhibited collagen-induced platelet aggregation: VBBPLA2 IC50 = 0.054, VLPLA2 IC50 = 0.072, NNOPLA2 IC50 = 0.814 μM. p-Bromophenacylbromide-inhibited sPLA2 had no inhibitory action on platelets. 36.17 μM VBBPLA2 completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA2s (~0.7 μM and ~7 μM towards cancer cells depended on both venom and cell type. VBBPLA2 (7.2 μM inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA2s exhibited no inhibitory effect towards LNCaP cells and some effect (8%–20% towards other cells. Thus, already sub-μM concentrations of sPLA2s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA2s and test cells, VBBPLA2 was the most growth inhibitory towards Bacillus subtilis and K-562 cells.

  13. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    Science.gov (United States)

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  14. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties

    Science.gov (United States)

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues. PMID:25970790

  15. Bacterial Communities: Interactions to Scale

    Science.gov (United States)

    Stubbendieck, Reed M.; Vargas-Bautista, Carol; Straight, Paul D.

    2016-01-01

    In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities. PMID:27551280

  16. Bacterial Communities: Interactions to Scale

    Directory of Open Access Journals (Sweden)

    Reed M. Stubbendieck

    2016-08-01

    Full Text Available In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities.

  17. The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

    OpenAIRE

    Andriamihaja, Mireille; Lan, Annaig; Beaumont, Martin; Audebert, Marc; Wong, Ximena; Yamada, Kana; Yin, Yulong; Tomé, Daniel; Carrasco Pozo, Catalina; Gotteland, Martin; Kong, Xiangfeng

    2015-01-01

    p-Cresol that is produced by the intestinal microbiota horn the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite On colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc(-/+) cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the 5 phase and with a slight increase of cell detachment without necrotic effect. At this...

  18. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    Science.gov (United States)

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. PMID:27109773

  19. Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.

    Science.gov (United States)

    Jensen, Nickels A; Gerth, Klaus; Grotjohann, Tim; Kapp, Dieter; Keck, Matthias; Niehaus, Karsten

    2009-03-10

    High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts. PMID:19111838

  20. The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae.

    Science.gov (United States)

    Krishnamurthy, Ajay; Kyd, Jennelle

    2014-08-01

    Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.

  1. The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells.

    Science.gov (United States)

    Andriamihaja, Mireille; Lan, Annaïg; Beaumont, Martin; Audebert, Marc; Wong, Ximena; Yamada, Kana; Yin, Yulong; Tomé, Daniel; Carrasco-Pozo, Catalina; Gotteland, Martin; Kong, Xiangfeng; Blachier, François

    2015-08-01

    p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc(-/+) cell line, we found that 0.8mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc(-/+) cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc(-/+) cells with 0.8mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc(-/+) and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their "respiratory reserve." At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes. PMID:25881551

  2. Mechanism of photocatalytic bacterial inactivation on TiO2 films involving cell-wall damage and lysis

    OpenAIRE

    C. Pulgarin; Kiwi, J.; Nadtochenko, V.

    2012-01-01

    This article addresses the cell wall damage of Escherichia coil (from now on E. coil) by TiO2 suspensions. The dynamics of TiO2 photocatalysis by thin films layers is described. The films were characterized by FTIR spectroscopy and atomic force microscopy (AFM). The E coil complete inactivation is shown to be due to the partial damage of the cell-wall components (peroxidation). A small increase in the cell wall disorder concomitant with a decrease of the cell wall functional groups leads to h...

  3. Immunotoxicity of nucleic acid reduced BioProtein - a bacterial derived single cell protein - in Wistar rats

    DEFF Research Database (Denmark)

    Mølck, Anne-marie; Poulsen, Morten; Christensen, Hanne Risager;

    2002-01-01

    NABP induced a humoral immune response and proliferation of phagocytic cell lines, mainly macrophages. The humoral response involved induction of NABP specific IgM and IgG. The proliferation of phagocytic cells involved increase of the white blood cell count of all dosed female groups. Males showed......, hyperplasia of Kupffer cells in the liver, increased granulopoiesis in spleen and bone marrow, and infiltration of lamina propria of the large intestine with eosinophilic granulocytes. The most consistent and pronounced changes were observed in the highest dose group, but even at the lowest dose level some...

  4. Bacterial chemoreceptors and chemoeffectors.

    Science.gov (United States)

    Bi, Shuangyu; Lai, Luhua

    2015-02-01

    Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on-off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.

  5. Mycobacterium tuberculosis PPE68 and Rv2626c genes contribute to the host cell necrosis and bacterial escape from macrophages.

    Science.gov (United States)

    Danelishvili, Lia; Everman, Jamie; Bermudez, Luiz E

    2016-01-01

    Alveolar macrophages are the main line of innate immune response against M. tuberculosis (Mtb) infection. However, these cells serve as the major intracellular niche for Mtb enhancing its survival, replication and, later on, cell-to-cell spread. Mtb-associated cytotoxicity of macrophages has been well documented, but limited information exists about mechanisms by which the pathogen induces cell necrosis. To identify virulence factors involved in the induction of necrosis, we screened 5,000 transposon mutants of Mtb for clones that failed to promote the host cell necrosis in a similar manner as the wild-type bacterium. Five Mtb mutants were identified as potential candidates inducing significantly lower levels of THP-1 cell damage in contrast to the H37Rv wild-type infection. Reduced levels of the cell damage by necrosis deficient mutants (NDMs) were also associated with delayed damage of mitochondrial membrane permeability when compared with the wild-type infection over time. Two knockout mutants of the Rv3873 gene, encoding a cell wall PPE68 protein of RD1 region, were identified out of 5 NDMs. Further investigation lead to the observation that PPE68 protein interacts and exports several unknown or known surface/secreted proteins, among them Rv2626c is associated with the host cell necrosis. When the Rv2626c gene is deleted from the genome of Mtb, the bacterium displays significantly less necrosis in THP-1 cells and, conversely, the overexpression of Rv2626c promotes the host cell necrosis at early time points of infections in contrast to the wild-type strain.

  6. An in vivo study of electrical charge distribution on the bacterial cell wall by atomic force microscopy in vibrating force mode

    Science.gov (United States)

    Marlière, Christian; Dhahri, Samia

    2015-05-01

    We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc.We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical

  7. Treatment of seafood processing wastewater using upflow microbial fuel cell for power generation and identification of bacterial community in anodic biofilm.

    Science.gov (United States)

    Jayashree, C; Tamilarasan, K; Rajkumar, M; Arulazhagan, P; Yogalakshmi, K N; Srikanth, M; Banu, J Rajesh

    2016-09-15

    Tubular upflow microbial fuel cell (MFC) utilizing sea food processing wastewater was evaluated for wastewater treatment efficiency and power generation. At an organic loading rate (OLR) of 0.6 g d(-1), the MFC accomplished total and soluble chemical oxygen demand (COD) removal of 83 and 95%, respectively. A maximum power density of 105 mW m(-2) (2.21 W m(-3)) was achieved at an OLR of 2.57 g d(-1). The predominant bacterial communities of anode biofilm were identified as RB1A (LC035455), RB1B (LC035456), RB1C (LC035457) and RB1E (LC035458). All the four strains belonged to genera Stenotrophomonas. The results of the study reaffirms that the seafood processing wastewater can be treated in an upflow MFC for simultaneous power generation and wastewater treatment. PMID:27254294

  8. Thermal Analysis of Whole Bacterial Cells Exposed to Potassium Permanganate Using Differential Scanning Calorimetry: a Biphasic Dose-Dependent Response to Stress

    Directory of Open Access Journals (Sweden)

    Marina K. Abuladze

    2009-01-01

    Full Text Available Differential scanning calorimetry (DSC was applied to estimate the impact of the toxic oxidant potassium permanganate (PM on the intracellular structural and functional alterations at whole cell level using soil bacteria Arthrobacter oxydans as a model culture. Differential scanning calorimetry (DSC was applied in order to estimate the impact of the toxic oxidant potassium permanganate (PM on the intracellular structural and functional alterations at the whole cell level using the soil bacteria Arthrobacter oxydans as a model culture. We compared the total melting heat and the temperature of DNA-protein complex (DNP melting at the PM application prior to the calorimetry measurement and after 24-h exposure at the concentration range 0.02–1.4 mM. The initial oxidative effect caused changes in the pattern of the whole cell melting spectra (mainly at the temperature range 56–78°C, the decrease of Tmax °C DNP melting, and did not influence significantly the total heat of bacterial melting at different concentrations of PM. The prolonged effect of permanganate up to 24 h was characterized by a biphasic dose-dependent response to stress estimated by the DSC technique and the colony-forming assay. The low doses of PM (0.02 and 0.2 mM stimulated cell proliferation, and increased the total whole cell melting heat and the temperature of DNP melting. The toxic effect of PM up to 0.04 mM reduced cell viability, changed the character of multipeaked thermograms, and lowered the total melting heat and the temperature of DNP melting in a concentration-dependent manner. This study presents the DSC method for evaluating and monitoring the effects of exposure to potential human and environmental toxicants.

  9. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    Science.gov (United States)

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

  10. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    Science.gov (United States)

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs. PMID:27091931

  11. SIV-induced Translocation of Bacterial Products in the Liver Mobilizes Myeloid Dendritic and Natural Killer Cells Associated With Liver Damage.

    Science.gov (United States)

    Evans, Tristan I; Li, Haiying; Schafer, Jamie L; Klatt, Nichole R; Hao, Xing-Pei; Traslavina, Ryan P; Estes, Jacob D; Brenchley, Jason M; Reeves, R Keith

    2016-02-01

    Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)-infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

  12. RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

    Directory of Open Access Journals (Sweden)

    Charles R. Cantor

    2011-03-01

    Full Text Available Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.

  13. Selective depletion of non-specific T cells as an early event in T cell response to bacterial and viral infections

    Institute of Scientific and Technical Information of China (English)

    JIANG Jiu

    2005-01-01

    @@ Early T cell depletion occurs prior to the development of an effective immune response to infections.Both antigen-specific and non-specific T cells are induced to express early activation markers soon after microbial infections.This is followed by massive depletion of non-specific T cells and extensive proliferation of antigen-specific T cells.Proliferating antigen-specific cells exhibit a broad spectrum of late activation markers while non-specific cells exhibit no sign of further activation